\. S PUBLIC HEALTH CHEMISTRY AND BACTERIOLOGY A HANDBOOK FOR D.P.H. STUDENTS DAVID McKAIL, M.D.(GLASG.), D.P.H. (CAMB.), F.R.F.P.S.G. Lecturer on Public Health and Forensic Medicine, St. Mungo's College, Glasgow ; Lecturer on Hygiene to Nurses, Glasgow Royal Infirmary ; Assistant Lecturer on School Hygiene to Teachers in Training, Glasgow Provincial Committee ; Examiner in Public Health for the D.P.H., Scottish Conjoint Board ; Part-time School Doctor, Glasgozv School Board mew Jtjorfe: WILLIAM WOOD AND COMPANY MDCCCCXII LIDflAlU' - PUBLIC HiALTH LIBRARY JOHN WRIGHT AND SONS LTD. PRINTERS, BRISTOL. PREFACE The present work is based on the notes prepared by the writer for his class while teaching Public Health Chemistry and Bacteriology. These were compiled from various standard works, and from articles appearing in the medical journals from time to time. No originality is therefore claimed for them, and grateful acknowledg- ment is made to the sources indicated.* The book is intended to assist in, and supplement, actual laboratory teaching, and not in any way to super- sede it. It has been the writer's endeavour therefore to make it as complete as possible, while leaving out matters which can be more satisfactorily taught and demonstrated than written about. No illustrations have been introduced, as the various instruments and apparatus are seen and used in the actual work. I desire to express my hearty appreciation of, and my best thanks for, the assistance received from a number * I am especially indebted to the following works : — Pakes' " Hygiene," Notter & Firth's " Hygiene," Moor & Partridge's " Aids to the Analysis of Foods and Drugs," Richter's " Organic Chemistry," the " Harmsworth Self -Educator," the " Encyclopaedia and Dictionary of Medicine and Surgery," Jordan's "Bacteriology," Muir & Ritchie's " Bacteriology," Hiss and Zinsser's " Bacteriology," Abel's " Laboratory Handbook," Stitt's " Practical Bacteriology," SimsWoodhead's "Bacteria and their Products," and the Lancet, Public Health, and the Bacteriological Supplement to the Medical Officer. 259680 iv PREFACE of friends, and very specially from Dr. R. M. Buchanan, Bacteriologist to the Corporation of Glasgow, and Dr. J. Hume Patterson, Bacteriologist to the County Council of Lanarkshire. To Dr. John A. Wilson, Assistant Bacteri- ologist, Glasgow, I owe a careful revision of the chapter on Special Bacteriological Examinations, including the Table on pp. 354—5, and much helpful criticism. To Dr. Archibald MacMillan I am indebted for criticism of some parts of the Chemical portion of the book, as a result of which certain changes have been made. DAVID McKAIL. Glasgow, 1912. CONTENTS PAGES Preface ------ iii-iv Introduction _.-... 1-4 Part I.— PUBLIC HEALTH CHEMISTRY. Chap. I. — Chemical Analysis - - - 5-18 Standard solutions, 6 ; Normal solutions, 7 ; Indicators, 10 ; Acidimetry and alkalinity, 12 ; Weighing and measuring, 14. Chap. II. — Water Analysis - - - 19-61 Introductory, 19 ; Collection of sample, 21 ; Physical examination, 22 ; Chemical examination, 22 ;] Quanti- tative estimation of lime, magnesia, phosphates and sulphates, 30 ; Hardness, 34 ; Organic matter, 38 ; Nitrites and nitrates, 47 ; Ice, 53 ; Mineral waters and aerated waters, 53 ; Interpretation of results, 53 ; Sewage and sewage effluents, 56. Chap. III. — Examination of Air - 62-70 Collection, 62 ; Odour, temperature, pressure and humidity, 63 ; Carbonic acid gas, 63 ; Ozone, 66 ; Oxid- izable and organic matter, 66 ; Noxious emanations, 67 ; Suspended matter, 69 ; Carbon monoxide, 69 ; Oxygen, 69 ; Gases in mines, 70. Chap. IV.— Soils - - -. - - 71, 72 Examination of sample : Ground air, 71 ; Ground water, 72 ; Soil temperature, 72. Chap. V.— Foods - - - - - 73-112 Milk, 73 ; Cream, condensed milk, infant foods, 84 ; Butter, 87 ; Cheese, 95 ; Cereals, 96 ; Starches, 101 ; Carbohydrates, 102 ; Golden syrup, honey, no ; Mustard, pepper, ginger, peas, meat extracts, 112. Chap. VI. — Beverages - 1 13-134 Coffee, 113; Tea, 115; Cocoa, 117; Lemon juice and lime juice, 119 ; Vinegar, 122 ; Beer, 125 ; Wine, 130 ; Spirits, 132. vi CONTENTS PAGES Chap. VII. — Disinfectants, Antiseptics, and Deodorants - 135-143 Bleaching powder, 135 ; Formalin, 136 ; Permanganate of potash, 136 ; Ferrous sulphate, 137 ; Carbolic acid, 137 ; Carbolic powders, 138 ; Sodium sulphite and sulphurous acid, 139 ; Zinc chloride, 139 ; Copper sulphate, 139 ; The Lancet-acetone-baryta method, 140. Chap. VIII. — Appendix - 144-147 Table of Glaisher's factors, 144 ; Weight of cubic foot of water vapour at various temperatures, 145 ; Alcohol tables, 145-147. Part II.— PUBLIC HEALTH BACTERIOLOGY. Introductory - 148-151 Chap. IX. — General Principles - - - 151-171 Bacteriological media, 151 ; Sterilization and disinfec- tion, 155 ; Cultural methods, 155 ; Modes of study, 156 ; Cultural reactions, 156 ; Liquefaction of gelatin, 159 ; Haemolysis, 160 ; Staining reactions and methods, 161 ; Polychrome stains, 167; Inoculation of animals, 169; Unicellular micro-organisms, 170. Chap. X. — Results of Bacterial Activity - 172-179 Products, 172 ; Infection, 173 ; Bacterial poisons, 175. Chap. XL — Immunity and Anaphylaxis - - 180-219 Immunity, 181 ; Immunity phenomena, 191 5 Theories of immunity, 198 ; Further immunity phenomena, 204 ; Anaphylaxis, 212. Chap. XII. — Micrococci - 220-229 Staphylococci, 220 ; Streptococci, 222 ; Pneumococcus, 224 ; Streptococcus mucosus, 226 ; Meningococcus, 226 ; Gonococcus, 227 ; Micrococcus tetragenus, Micrococcus catarrhalis, Micrococcus melitensis, 228. Chap. XIII. — Non-sporing Bacilli - - 230-303 The Colon-typhoid-dysentery group, 230 ; Capsulated bacilli, 240 ; Bacillus acidi lactici (Hueppe), 242 ; Minute bacilli, 243 ; Morax-Axenfeld diplo-bacillus, 245 ; Diphtheria bacillus, 246 ; Bacillus mallei, 250 ; Bacillus pestis, 254 ; Summary of Lancet reports of plague in China, 1910-11, 263 ; Tubercle bacillus, 273 ; Other acid-fast bacilli, 284 ; Actinomycosis, or Ray fungus disease, 286 ; Summary of final report of British Royal Commission on Tuberculosis, 288. CONTENTS vii PAGES Chap. XIV. — Sporing Bacilli - - - 304-320 Spore-bearing aerobic bacilli, 304 ; Spore-bearing anaerobic bacilli, 310 Chap. XV. — Spirilla - 321-325 Spirillum choleras Asiaticae, 321 ; Spirilla other than the cholera spirillum, 324. Chap. XVI. — Spirochetes - 326-329 Spirochaeta recurrentis, 326 ; Spirochagta Vincenti, 327 ; The micro-organism of Syphilis, 327 ; Yaws, 329. Chap. XVII. — Yeasts and Moulds - - 330-344 Yeasts, 331 ; Moulds, 334. Chap. XVIII. — Special Bacteriological Examina- tions - 345-383 Water, 345 ; Air, 363 ; Soil, 365 ; Dust, sewage and sewage effluents, and excremental matters, 365 ; Milk, 366 ; Butter, Cheese, Shellfish, Watercress and other vegetables, 376 ; Disinfectants, 377. Appendix __..-- 384-392 Regulations for the Diploma in Public Health, 384 ; Preservatives in milk and cream, 388 ; Bovine and human types of Tubercle bacilli, 390. Index - • 393 Addenda and Erratum Page 38, line 7 : after ' ' present " add " as chloride, sulphate, etc." Page 79, Preservatives in Milk : See new Regulations (1912) on page 388. Page 80, line 30 : after ' ' tubes " add " plus a drop of phenolphthalein solution " Page 84, Preservatives in Cream : See new Regulations (1912) on page 388. Page 233, Antityphoid vaccine : Leishman sterilizes the typhoid culture at 53° C for one hour* Page 277, line 2 from foot : after " growth " add " on glycerin egg medium (see page 391) " Page 257, line 5 from foot : for " Limond," read " Simond " PUBLIC HEALTH CHEMISTRY AND BACTERIOLOGY. INTRODUCTORY. pUBLIC HEALTH Chemistry and Bacteriology do not r-*- differ fundamentally from general chemistry and bacteriology, and in fact are based on these subjects, of which they are specialized departments. The same principles underlie the part as the whole, and the accumu- lation of scraps of knowledge derived from the parent sciences, under the heading of Public Health Chemistry and Bacteriology, is justifiable only on the score of con- venience and the importance of economizing the student's time. It is therefore necessary to remember that the whole is greater than the part, and that to have wide and luminous views of the subjects so designated, the study of them should not be strictly utilitarian, but be extended in all necessary directions as much as possible. A knowledge of Public Health Chemistry and Bacteri- ology has become more urgently called for, owing to the increase of Public Health work, to participate in which it is necessary to possess a Diploma in Public Health. The General Medical Council at their meeting on ist December, 191 1, adopted in an amended form the resolu- tions and rules which form the Regulations for the Diploma in Public Health. These are printed in full in an Appendix to this volume. For our present purpose it is sufficient to quote here Rule 2. Rule 2. Every Candidate for a Diploma in Sanitary Science, Public Health, or State Medicine shall have produced satisfactory evidence that, after obtaining a registrable qualification, which should be registered before admission to examination for the diploma, he has received practical instruction in a laboratory or 2 INTRODUCTORY laboratories, British or foreign, approved by the licensing body granting the diploma, [in which chemistry, bacteriology, and the pathology of the diseases of animals transmissible to man are taught. Note. — The laboratory instruction shall cover a period of not less than four calendar months, and the candidate shall produce evidence that he has worked in the laboratory for at least 240 hours, of which not more than one-half shall be devoted to practical chemistry. The laboratory course should be so arranged as to lay special stress on work which bears most directly on the duties of a medical officer of health. The amendments in this rule reduce the number of months of instruction from six to four, and define the hours worked as 240, of which at least 120 shall be devoted to Bacteriology. The net result is that students will thus require to do as a minimum 15 hours' practical laboratory work per week for four months. Many students will find it more convenient to do 12 hours per week for a so-called six months' term of 20 weeks. The final clause of the rule, namely, " The laboratory course should be so arranged as to lay special stress on work which bears most directly on the duties of a medical officer of health " might usefully have been made more specific, and been extended to include the phrase, " and the Examiners for the Diploma shall have special regard to this recommendation." The student will "be well advised to exceed the minimum periods above laid down, as far as he can, and more espe- cially if he is not already well informed in the subjects of chemistry and bacteriology. The scope of the work and the methods may be thus summarized on the basis of the Syllabus of the Scottish Conjoint Board : — DIPLOMA IN PUBLIC HEALTH. Public Health Laboratory Work Course comprises : — Physical, Chemical, and Bacteriological Examination of water, sewage and sewage effluents, air and other gases, food stuffs, beverages, soils, and building materials. Examination of disinfectants, antiseptics, deodorants. INTRODUCTORY 3 Detection of Poisons in foods, dress, decoration. Examination of parasites and other animal organisms found in the body and human food stuffs. Bacteriology and Bacteriological Methods : apparatus, media, modes of culture ; culture and recognition of the principal pathogenic organisms ; bacteriological examina- tion of water, air, and foods ; antitoxins ; principles of serumtherapy and immunization ; cultivation and recognition of micro-organisms in relation to epidemic and other diseases. Modes of Examination. Physical. — Inspection by the unaided senses — colour, odour, taste, transparency, turbidity, etc. ; specific gravity ; microscopic examination ; spectroscopic examin- ation ; polariscopic examination. Chemical. — Examination for proximate principles, e.g., water, solids, fat, sugar, etc. ; search for and identification of chemical impurities such as ammonia, lead, borax, etc. ; quantitative estimations of above. Bacteriological. — Enumeration of bacteria present ; search for pathogenic forms ; if found, isolation and identification. Part I. PUBLIC HEALTH CHEMISTRY. IT is necessary, in the first place, to consider carefully certain points of chemical practice, which form the basis of much of the subsequent work, and which must be thoroughly understood to enable the latter to be readily followed and apprehended. CHAPTER I, CHEMICAL ANALYSIS. This is of two kinds — Qualitative or Quantitative. 1. Qualitative. — Consists in proving the presence or absence of certain metals or salts, or generally of chemical elements or radicles or compounds in a substance or solution, by the use of a series of tests. 2. Quantitative. — Consists in separating out the con- stituents of any composite body and accurately estimating the amount of each of them. This may be done in three ways : Gravi metrically, Volumetrically, Colorimetrically. Gravimetric method. — The desired constituent is separated out in a known form, and this is accurately weighed. As a method it is often very complicated, very lengthy, and requires elaborate apparatus and much skill. Volumetric method. — This consists in submitting the substance to certain characteristic reactions, a measured quantity of a solution of known strength being added until a change looked for occurs. From the quantity of reagent used, the amount of the substance found can be calculated by known chemical laws. It is less elaborate, much more quickly accomplished, needs simpler apparatus as a rule, is susceptible of great accuracy, and the skill required is less specialized. Colorimetric method. — This consists in using a reaction which produces a coloured tint, which is compared with the tint obtained from the same treatment of a known G PUBLIC HEALTH CHEMISTRY quantity of the substance under investigation. Exact matching of the tints is arrived at, either by dilution of the stronger, or by putting up several standard tints. Requirements for Volumetric Analysis. — i. Solution of reagent or test, the chemical power of which is accurately known ; this is called the Standard Solution. 2. A graduated vessel, from which portions of the standard solution may be accurately delivered : the Burette. 3. Some indication, unmistakeable to the eye, that the reaction is terminated or concluded : the Indicator. The process is called, a titration, i.e., an estimation of the titre or strength of a solution, and the person is said to titrate the solution. Volumetric methods may be classified thus : — 1. Neutralization of acids by alkalies, and vice versa — acidimetry and alkalimetry. 2. Reduction or oxidation of substances — for example, ferrous sulphate titrated with potassium permanganate illustrates both actions. 3. Precipitation of some insoluble and definite com- bination— for example, precipitation of AgCl in testing for chlorides. STANDARD SOLUTIONS. A standard solution of any substance is made when a known quantity of that substance is dissolved in a known quantity of water. Then the strength can be expressed definitely as — So many pounds per gallon, or pint, or ounce ; or grains „ „ „ or „ grammes per litre or cubic centimetre (c.c.) ; or „ milligrammes ,, „ ,, Thus we might have a standard solution of NaCl of these strengths : (1 gallon = 10 lbs. = 70,000 grs.). 1 lb. per gallon ; then 1 fluid grain = o-i grain NaCl. 1 drachm per fluid ounce ; then 1 minim = 0-125 gr- NaCl. 1 gramme per litre ; then 1 cubic centimetre — o-ooi gramme, or 1 milligramme of NaCl per c.c. CHEMICAL ANALYSIS 7 The metric system of weights and measures is found to be very convenient for such solutions, because — i gramme dissolved in i litre = i milligramme in i c.c. ; or n grammes dissolved in i litre = n milligrammes in i c.c. Standard solutions often have their strength expressed in terms of some other substance which they measure — usually an elementary substance. For example — AgN08 + NaCl = AgCl + NaN03 170 (23 + 35-5). From this equation we see that 170 parts of silver nitrate precipitate completely 58-5 parts of sodium chloride containing 35-5 parts of chlorine. If we wish to estimate the amount of CI present in a solution of unknown strength, we can titrate with a solution of silver nitrate of known strength — that is, a standard solution. What strength shall we make it ? a. 170 grm. AgN03 in 1 litre of water will precipitate 35-5 grm. CI ; then 1 c.c. will precipitate 35-5 mgr. CI. b. 17-0 grm. AgN03 in 1 litre of water will precipitate 3-55 grm. CI ; then 1 c.c, will precipitate 3"55 mgr. CI. c. J— = 4-78 grm. in 1 litre of water will precipitate 1 grm. CI; then 1 c.c. will precipitate 1 mgr. CI. The strength chosen depends on — (1) The simplicity desired ; (2) The strength of solution to be tested ; (3) Whether any one of these strengths will be more useful than the others for other estimations, and so save needless duplication of solutions. NORMAL SOLUTIONS. These are standard solutions made to a certain strength on the basis of chemical theory and practice. Thus, from the equation — NaOH + HC1 = NaCl + H20 40 36-5 we see that 40 parts of sodium hydrate are exactly neutral- ized by 36-5 parts of hydrochloric acid. If therefore we 8 PUBLIC HEALTH CHEMISTRY make a standard solution of NaOH 40 grammes to 1 litre, and one of HC1 36-5 grammes to 1 litre, then — 1 c.c. stand, sol. NaOH = 1 c.c. stand, sol. HC1, and if we can build other solutions on the same plan, we shall have a whole range of solutions which are chemically equivalent c.c. for c.c. This will obviate having a strength of acid for titrating soda, another strength for potash, another for baryta, and so on, and reversely. This result is obtained, or rather attained, by using normal solutions, which are thus defined : — A normal solution is one which contains in 1 litre of distilled water at 160 C. the hydrogen equivalent of the active reagent weighed in grammes, hydrogen being taken as one gramme. Such a solution is usually indicated by N the mark N or — . If diluted, the degree of dilution is indicated by a denominator, thus — N JN JN_ _N_ 2 10 100 1000 which are respectively— seminormal decinormal centinormal millinormal The hydrogen equivalent of a substance is found by taking its molecular weight, the number of atoms usually replaced by hydrogen, and .the valency of these atoms. Divide the molecular weight by the product of the number of atoms and their valency. Thus — NaCl m.w. =58-5 no. of replaceable atoms=i valency=i ; therefore — N/i NaCl=58«5/ix 1=58-5 grm. to 1 litre of water. Similarly — HC1 m.w.=36-5 n.r.a.=i v.=i; hence N/i=36-5 grm. per litre. H2S04 m.w. = 98 grm. N/i = 49 grm. per litre. HNO3 m.w. = 63 „ N/i = 63 ' „ NaOH m.w. = 40 ,, N/i = 40 ,, KOH m.w. = 56 „ N/i = 56. ;, Na2C03 m.w. = 106 ,, N/i=-53 K2C03 m.w. a 138 „ N/i = 69 CHEMICAL ANALYSIS 9 Where the substance possesses molecules of water of crystallization, the weight of these must be added to the sum of the molecule proper, in order to arrive at the right figure for the normal solution. Thus — H2C204+2H20 m.w. = i26 N/i=63 grm. per litre (oxalic acid). H3C6H507+H20 m.w.=2io N/i=70 grm. per litre (citric acid). H2C4H406 m.w. = i5o N/i= 75 grm. per litre (tartaric acid). HC 2H 30 2 m.w. =60 N /i =60 grm. per litre (acetic acid). Some salts with enlarged molecules : — Ferrous sulphate, FeS04+7 H20 molecular weight =278 Copper sulphate, CuSOd-|- 5 H20 „ „ =249 Lead acetate, Pb(C 2H 30 2) 2+3 H 20 ,, „ =379 Sodium thiosulphate, Na2S20 3+5. H20 ,, ,, =248 The hydrogen equivalent of some reagents is not so easily arrived at ; for instance, potassium permanganate, variously written as K2Mn208 and KMn04. The molecular weight of the first formula is 316, and of the second is 158. Nevertheless, the normal solution is not 158 grammes per litre, but is 31-6 grammes per litre. This is because potassium permanganate does not react — as its formula might suggest — as a manganate, but reacts as a double salt of potassium and manganese, as if it were written thus — K20 + Mn207, and in reaction the latter oxide is reduced and oxj^gen liberated ; thus — K 2Mn 20 8+3 H 2SO 4=K 2SO 4+2 MnSO 4+3 H 20+5 O 316 80 We here see that 316 grammes of permanganate of potash liberate 80 grammes of oxygen, which are chemically equivalent to 10 grammes of hydrogen, and so 31-6 10 PUBLIC HEALTH CHEMISTRY grammes of the salt are equivalent to I gramme of hydrogen. Hence — N/i KMn04 = 31-6 grm. per litre. N/io do. = 3-16 do. do. N/ioo do. ^ -316 grm. do. Non- Standardized Solutions : — Ammonia free water. Organically pure ammonia free water. Nessler's solution. Methyl-orange solution (1 grm. per litre of water). Phenolphthalein solution (1 % in 50 % alcohol). Starch solution (5 grm. per litre of boiling water). Baryta water (5 grm. of crystallized barium hydrate in 1 litre of freshly boiled distilled water. Stopper and set aside for three days, and decant off clear liquid. About Metaphenylene - diamine (5 grm. per litre aq. dest. slightly acidulated with a few drops of sulphuric acid). Naphthylamine acetate in sulphanilic acetate : — a. 3 to 4 grm. of sulphanilic acid dissolved in 1 litre of dilute acetic acid. b. frds grm. of naphthylamine are boiled with 150 c.c. of aq. dest., the colourless liquid poured off and diluted to 1 litre with dilute acetic acid. c. Mix equal bulks as required for testing. Phenol-sulphonic acid (32 c.c. of pure concentrated sulphuric acid are added to 4 c.c. of pure phenol. Heat to ioo° C. for 2 to 3 hours. Cool, and add no c.c. of distilled water). INDICATORS. These may be classified under two heads : — (1) Neutrality indicators, which give a special reaction with acid or alkaline liquids, or with both ; (2) All others, such as starch, iodine, chromate of potash, permanganate of potash, and soap lather. Neutrality indicators are divided thus by R. T. Thompson : — CHEMICAL ANALYSIS 11 (i) Methyl-orange group : are most susceptible to alkalies ; methyl-orange, lacmoid, cochineal, congo-red ; (2) Phenolphthalein group : are most susceptible to acids ; phenolphthalein, turmeric ; (3) Litmus group : are inter- mediate in susceptibility — litmus, rosolic acid, phenace- tolin. Sensitiveness. — Phenolphthalein, lacmoid, rosolic acid, and phenacetolin showed change of colour with one-fifth quantity of acid or alkali required by methyl-orange and litmus ; that is to say, if the two latter required in 100 c.c. of acid or alkali 0-5 c.c. to show change of colour, the former required only o-i c.c. Neutral point of one indicator does not coincide exactly with that of other indicators. Thus, saliva is generally neutral to litmus, alkaline to lacmoid or congo-red, and acid to turmeric. Fresh milk shows similar variations. 1. Litmus solution is violet coloured. Acids change it to red ; alkalies to blue. In cold solution it may be used for the titration of — Hydrates of soda, potash, ammonia, lime, baryta, etc. Nitric, sulphuric, hydrochloric, and oxalic acids. Arsenites and silicates of soda and potash. In boiling solution — Carbonates and bicarbonates of K, Na, Ca, Mg, Ba. Sulphides of sodium and potassium. 2. Methyl-orange is orange-brown in colour. Acids change it to pink ; alkalies to faint yellow. Only used in cold solution, and then for titration of — Hydrates, carbonates, bicarbonates of K, Na, NH3, Ca, Mg, Ba, etc. Sulphides, arsenites, silicates, borates of K, Na, NH3, Ca, Mg, Ba, etc. All the mineral acids. Sulphites. Half the base in the alkaline and earthy alkaline phosphates and arseniates. Not for organic acids, nor in presence of nitrous acid or nitrites, which decompose it. 12 PUBLIC HEALTH CHEMISTRY 3. Phenolphthalein is colourless in solution. Acids cause no change in colour ; alkalies change it to purple-red. Used in the cold for titration of — Alkaline hydrates, except ammonia. Mineral acids. Organic acids (oxalic, tartaric, acetic, citric, and others). Carbonates to bicarbonates. May be used in alcoholic solutions, and hence for organic acids insoluble in water. Also for acids combined with bases, like morphia, quinine, brucine, etc., the organic base having no effect on it. 4. Rosolic Acid is pale yellow in solution. With acids, unchanged ; with alkalies, violet-red. Good for mineral acids and oxalic. Not reliable for organic acids. 5. Turmeric, yellow in colour. With acids, bright yellow ; with alkalies, reddish-brown. 6 Lacmoid, blue and red papers are best form for use. These are an excellent substitute for methyl-orange when latter cannot be used. It is a derivative of resorcin, and is allied to litmus. The other indicators will be alluded to as their use is required. Rules as to use of indicators commonly employed : — Methyl-orange for mineral acids. Phenolphthalein for organic acids. Litmus for organic acids in presence of free CO 2 (e.g. in beer). ALKALIMETRY AND ACIDIMETRY. Perform the following exercises : — 1. Titrate 1 c.c. 50 per cent NaOH diluted with a little water (distilled) with N/i H2S04, using a few drops of litmus as indicator. Take the NaOH in a porcelain basin— add the water and the litmus solution. Then take a burette and fill it with the normal acid solution ; be careful that the nozzle is CHEMICAL ANALYSIS 13 full, and that the lowest part of the meniscus is opposite the zero mark. Now add the acid I c.c. at a time, stirring after each addition, with a glass rod. Continue the process until the colour of the solution changes to red. The result will be accurate to I c.c. With practice the end reaction can be judged more accurately ; but, as already mentioned, a certain quantity of acid is required for change of colour, and this is greater with litmus than with some other indicators. Say that 13 c.c. of N/i H2S04 were required. How much NaOH was present in the 1 c.c. of sample ? 1 c.c. N/i H2S04 =1 c.c. N/i NaOH But N/i NaOH = 40 grm. per litre Then 1 c.c. N/i NaOH = 0-040 grm. Hence 1 c.c. N/i H2S04 = 0-040 grm. NaOH Then 13 c.c. ,, ,, = 0-52 grm. NaOH. But 13 c.c. were required to neutralize 1 c.c. of sample. Therefore 1 c.c. of sample contains o#52 grm. NaOH, or 100 c.c. ,, will contain 52 grm. NaOH. The discrepancy between 50 grm. in 100 c.c. and 52 grm. may be due to : (1) Inaccuracy in making the soda solution originally ; (2) Inaccuracy in measuring the 1 c.c. of soda solution ; (3) Inaccuracy in the strength of the normal acid solution ; (4) Inaccuracy in titration. 2. Repeat the experiment, using 1 drop methyl-orange as indicator. 3. Repeat the experiment, using 1 drop phenolphthalein as indicator. 4. Titrate 5 c.c. 25 per cent H 2SO 4 diluted with a little water, with N/i NaOH, using a few drops of litmus as indicator. 5. Repeat the experiment, using 1 drop methyl-orange as indicator. 6. Repeat the experiment, using 1 drop phenolphthalein as indicator. 14 PUBLIC HEALTH CHEMISTRY Calculate out strength of solution by method similar to above. Thus, say that 25 c.c. of N/i soda are required to neutralize 5 c.c. of sample, said to be 25 per cent sulphuric acid. Then 1 c.c. N/i soda = or is chemically equivalent to 1 c.c. N/i sulphuric acid. But N/i H 2SO 4 sat 98/2 or 4.9 grm. per litre. Hence 1 c.c. „ „ = 0-049 grm. H2S04 Hence 1 c.c. N/i NaOH = 0-049 &rm- H 2SO 4 And 25 c.c. „ „ = 25 X 0-049 = I,225 grm- H2S04. But 25 c.c. N/i NaOH were required to neutralize 5 c.c. of sample ; therefore 5 c.c. of sample contain 1-225 grm- of sulphuric acid, and 100 c.c. of sample contain 24-5 grm. of sulphuiic acid ; that is, if 100 c.c. are taken as 100 grm., 24*5 per cent. The sources of error are as before. WEIGHING AND MEASURING. Measuring of Solutions. — Small quantities, like 1 c.c, 2 c.c, etc, up to 25 c.c. or 50 c.c, are most accurately measured by pipette, or on some occasions by burette. For larger amounts, like 100 c.c, 250 c.c, 500 c.c, and 1000 c.c, flasks with a narrow neck and a mark thereon are the best. When extreme accuracy is not essential, the ordinary graduated measure is quite efficient. Be careful in pipet- ting certain liquids not to get any drawn into the mouth. Never pipette strong sulphuric acid or ammonia by mouth suction. On Weighing with the Chemical Balance. — To weigh a certain quantity of a substance the necessary weights are placed in the right-hand pan of the balance. Some of the substance is then placed in the left-hand pan, or preferably in a watch-glass of known weight, or balanced by a similar one in the other pan. The handle or screw is now turned, and the balance put in action. If the pointer swings more on the side away irom the weights, that is more to the left side, the amount of substance is too little. The handle must now be turned down, and the balance thus placed CHEMICAL ANALYSIS 15 at rest , before any further manipulations are tried. There- after more of the substance is added, and the same tech- nique carefully observed. To obtain the weight of a substance or dish, or both, these are placed in the left-hand pan, and a trial weight is put on the right pan. If this is too much, the next lower weight is tried, and so on, being careful to observe the technique outlined above. The weight is read from the vacant spaces in the case, and checked on removing the weights. Always use a watch-glass in weighing a salt or substance not contained in a vessel of some kind. Atomic Weights of the elements the recently assigned. Aluminium Arsenic Barium Boron Bromine Calcium Carbon Chlorine Copper Hydrogen . . Iodine Iron Lead Magnesium . Manganese. . Mercury Nitrogen . . Oxygen Phosphorus Potassium Silver Sodium Sulphur Tin Zinc — The following table gives for some older atomic weight, and that more OLDER ATOMIC WEIGHT. NEWER 27 26'9 75 74*4 137 136-4 11 10-9 80 79-4 40 397 12 n-9 35*5 35*1 63 63-1 1 — 127 126-0 56 55-5 207 206-4 24 24-18 55 54'52 200 198-5 14 i3'9 16 i5'9 31 30-8 39 38-8 108 107-1 23 22-9 32 3i-8 118 ii8-i .. . 65 64-9 16 PUBLIC HEALTH CHEMISTRY Volume and Density of Gases.— All gaseous molecules, at the same temperature and pressure, occupy the same volume. This is another way of stating Avogadro's law, that " equal volumes of all gases (at a temperature suffi- ciently remote from their condensation-point — the so-called permanent gases) at the same temperature and pressure, contain the same number of molecules." From this it follows, that if we know the volume occupied by one gaseous molecule at standard temperature and pressure, we thereby know the volume of all gaseous molecules at the same temperature and pressure. The volume occupied by the molecular weight of hydrogen, expressed as grammes, that is 2 grammes of hydrogen gas at 0° C. and 760 mm. pressure, is 22-32 litres. Hence the statement, " the molecular weight of- any gas, expressed in grammes, measures 22-32 litres, at standard temperature and pressure, that is, 0° C. and 760 mm., or 320 F. and 29-9 inches of mercury." The Crith. — One litre of hydrogen gas at standard temperature and pressure weighs 0-0896 gramme. This weight has been called a crith. It follows from the above statement of Avogadro's law, that one litre of oxygen gas will contain the same number of molecules, each one 16 times the weight of the hydrogen molecule (as 32 : 2), and hence the weight of one litre of oxygen gas will be 16 criths, or 16 x 0-0896 grm. Similarly, one litre of carbon dioxide gas weighs 22 criths (as 44 : 2). The weight, there- fore, of one litre of an elementary gas (with exceptions) is equal to its atomic weight in criths ; and of one litre of a compound gas, is equal to half its molecular weight in criths. Metric System. The weights and measures of the metric system are those used nowadays in most Public Health work, although statements like " grains per gallon " still persist. The chief units employed are the following : — Length. — 1 metre = the length of a rod of platinum at the temperature of me] ting ice. This rod is kept at Paris, with official copies in the large towns. Equals 39-37 inches. CHEMICAL ANALYSIS 17 decimetre = ^ of a metre. centimetre TTRT of a metre, millimetre = Woo °* a metre, micron (/u) == toVtf °* a millimetre, kilometre = iooo metres. Mass. — gramme = the mass of i cubic centimetre of distilled water at the temperature of its maximum density, 40 C. or 39*2° F., 1 c.c. at 160 C. or 6o*8° F. = 0*9989 gramme. 1 gramme equals 15*432 grains, decigramme = -fy of a gramme, centigramme = ^ of a gramme, milligramme = y^Vo- °f a gramme, kilogramme = 1000 grammes. Volume. — litre 5= the volume or capacity of 1 kilogramme of distilled water at 40 C. Equals 35*196 imperial fluid ounces, decilitre = centilitre = millilitre -^ of a litre. -1- of a litre. irnr Ttftnr of a litre- cubic centimetre = TTjVo °^ a litre. Factors for Conversion from One Scale to the Other. Grammes into grains ,, into ounces, avoirdupois Kilogrammes into pounds Grains into grammes Avoirdupois ounces into grammes Troy ounces into grammes Cubic centimetres into fluid ounces, impl Litres into fluid ounces, imperial Fluid ounces into cubic centimetres Pints into litres Metres into inches Inches into metres The following tables give metric equiv measures of mass and capacity :— . X 15-432 . X 0*03527 . ' X 2*2046 X 0*0648 . X 28*35 . X 31*104 1. X 0-0352 . X 35'2 . X 28*42 . X 0*568 . . X 39"37 . X 0*0254 alents of imperial J 8 PUBLIC HEALTH CHEMISTRY Length. — i mm. (millimetre) = ^ of an inch, i cm. (centimetre) = f of an inch, i inch = 25*4 millimetres or 2 J centimetres. Mass. — 1 mgr. (milligramme) = 0-01543 grain (or approx. fc gr.). rgrm. (gramme) = i5'4323 grains. 1 kgr. (* kilo " or kilogramme) = 2 lb. 3 \ oz. avoirdupois. 1 pound avoirdupois = 453'592 grammes. 1 ounce avoirdupois = 28-35 grammes. 1 grain = 0*0648 gramme or 64*8 milligrammes. Capacity. — 1 centimil = 0*17 minims (approx.) imperial measure. 1 decimil = 1*7 minims (approx.) imperial measure. 1 c.c. (cubic centimetre) (or 1 mil) = 16*9 minims, imperial 1 '■-'■■'■ measure. •; 1 L. (litre) =35-196 fluid ounces (35 fl. oz., 1 fl. dr., 34 min.), imperial measure. 1 fl. ounce, imperial measure = 28-42 cubic centimetres i pint, imperial measure = 568-34 cubic centimetres. 1 gallon, imperial measure = 4*546 litres, or 10 lb. avoir- dupois of pure water at 620 F., and under an atmos- pheric pressure of 30 inches of mercury. CHAPTER II. WATER ANALYSIS. The examination of water samples is most commonly made to determine the presence or absence of evidence of sewage pollution. If the pollution is gross, the evidence of the unaided senses will cause its rejection. Few people would use for domestic purposes water which was turbid, or contained suspended matter, or had a peculiar colour, or smelt badly, or had a disagreeable taste. Yet a water may have none of these characteristics, and still be dangerous or unfit for domestic use. Chemical analysis is then often of service in distinguishing good from bad waters. Except in the case of a poisonous metal, the analysis does not aim at finding things deleterious in them- selves, but the search is made for constituents which suggest- the presence of deleterious or dangerous substances. In the case of sewage pollution, the latter are micro- organisms, and the former are those constituents of sewage which are readily detected, namely, chlorides from the urine, ammonia from the urea, and so-called albuminoid ammonia from any albuminous matter. As the average adult excretes 6 to 9 grammes of chlorine daily as chlorides, and 1 part of chlorine per 100,000 parts of water is easily estimated, the pollution produced by one day's excretion of urine into a water would thus be inferable from a rise of the chlorine content 1 per 100,000 even where the dilu- tion was into 600-900 litres or 120-180 gallons of water. The ammonia estimation is much more sensitive, a rise of 1 part in 50,000,000 being detectable. The urea excretion of an adult averages 33 grammes per day, and by the influ- ence of the micrococcus ureae this is changed to ammonium carbonate, thus : — CO(NH 2) 2 + 2H 20 = CO 3(NH 4) 2 60 96 Hence, if 60 grammes of urea give rise to 96 grammes of ammonium carbonate, containing 34 grammes of ammonia, 33 grammes of urea will give rise to the formation of 187 20 PUBLIC HEALTH CHEMISTRY grammes of ammonia, which diluted into 900,000 litres or 200,000 gallons of water, would still have caused an appreci- able rise on the amount (if any) of ammonia already present. This illustrates the delicacy of some of these tests. Various other estimations are carried out, such as, to determine the presence or absence of poisonous metals, the degree of hardness, etc. These are usefully sum- marized thus : — Water Analysis. This consists of three parts : — 1. Physical examination. 2. Chemical examination. 3. Bacteriological examination. Physical Examination — Transparency — clear and bright — turbid. Suspended matter — stand for twenty-four hours in glass with conical bottom. Colour — two-foot tube. Taste — uncertain — iron detectable in I gr. per gallon — NaCl in 75 grains per gallon. Smell. Microscopic characters of sediment — Particles of animal, vegetable, and mineral origin. Micro-organisms, bacterial and protozoal. Chemical Examination. — Reaction. Dissolved solids — Total. Fixed. Volatile. Charring on ignition — fumes — odour. Chlorine. Poisonous metals — Pb, Cu, Fe, Zn, As, Sn. Lime and magnesia. Phosphates and sulphates. Free carbonic acid — bicarbonates — carbonates ; dissolved oxygen ; sulphuretted hydrogen. Hardness — Total. Permanent. Temporary. WATER ANALYSIS 21 Free and saline ammonia. Albuminoid ammonia. Oxygen absorption. Nitrates. Nitrites. Bacteriological Examination. — (a) Absolute minimum. 1. Enumeration of bacteria growing in a medium at air temperature i8°-22° C. 2. Search for Bacillus coli. If found — identification and enumeration. (b) Additional. 3. Enumeration of bacteria growing at blood heat 370 C. 4. Search for and enumeration of streptococci. 5. Search for Bacillus enteritidis sporogenes. (c) Special procedures. 6. Isolation of Bacillus typhosus from water. 7. Isolation of Spirillum cholerae. COLLECTION OF SAMPLE. A fair average sample should be taken in a clean glass vessel with a glass stopper. In filling the vessel from a pond, lake, reservoir, or river, the mouth of it should be sunk two inches below the surface, and the vessel should be filled and emptied once or twice. If a surface specimen is wanted, then of course the sample will be so taken. When sampling water from a pipe or tap, unless the effect of the water on the pipes is under examination, the water should be allowed to run to waste for a few minutes before filling the vessel. The stopper should be tied in but not sealed. A convenient receiver is a Winchester quart bottle which holds half a gallon, and this is a suitable quantity for the usual analysis. Along with the sample, a written statement should be sent, giving full particulars as to mode of collection, place, time, recent meteorological conditions, reason why analysis is desired, etc., etc. 22 PUBLIC HEALTH CHEMISTRY The examination should be undertaken as soon as pos- sible, since changes take place on keeping. If delay is unavoidable, changes should be kept at a minimum by packing in ice. PHYSICAL EXAMINATION. Transparency. Suspended matter. Colour. — Best, bluish or greyish ; greenish, from algae ; yellow or brown suspicious, except peaty. Taste. Smell. — Place 250 c.c. in a glass-stoppered bottle. Put on water-oven at 300 C. for a few minutes. Remove stopper, and smell at once. Sediment. — Let water stand for a few hours, pipette a few c.c. from bottom, centrifuge, mount a drop on a clean slide, and examine. The deposit may contain a very large number of things. 1. Mineral matter, such as sand, clay, etc. 2. Vegetable matter : (a) Living — such as bacteria, yeasts, moulds, diatoms, desmids, rotiferae ; (b) Dead — vegetable cells, husks of grain, cotton or linen fibres, starch granules. 3. Animal matter : (a) Living — such as ova, insects, worms, etc. ; (b) Dead — such as hairs, scales, muscle fibre. CHEMICAL EXAMINATION. Reaction. — Most drinking waters are alkaline in reac- tion. Upland surface water is often acid from humic and ulmic acids ; and this is important, as these acids dissolve lead. Sewage-contaminated waters usually retain their alkalinity. Waters polluted by refuse from chemical or dye works are sometimes acid in reaction. Dissolved Solids. — The suspended matter is usually allowed to settle before testing for the solids in solution. The latter are estimated as total, fixed, and volatile. Also note, -when igniting dried solids, presence or absence of fumes, odour, and charring. Total Solids. — (1) Take a weighed platinum or porcelain dish of sufficient size ; (2) Add 100, 200, 250, 500, or WATER ANALYSIS 23 iooo c.c. of water sample ; (3) Reduce bulk by moderate heat, avoiding boiling or spurting. Or, to a small dish successive quantities of the sample are added, a note being kept of the amount ; (4) Evaporate to complete dryness at ioo° C. (2120 F.) on water-bath ; (5) Now place in water-oven at ioo° C. for half an hour to remove all traces of moisture. Some analysts advise this drying to be done at 1050 C. in hot-air chamber ; (6) Cool in dessicator ; (7) Weigh. To make quite sure that residue is dry, items 5, 6, and 7 can be repeated until weight is constant ; (8) Subtract weight of dish ; difference is weight of total residue in amount of sample taken ; (9) Calculate as parts per 100,000, and as grains per gallon. Fixed Solids. — (1) Incinerate the dried solids at as low a heat as possible ; (2) Watch the process for blackening or charring, fumes or odour. A piece of dry starch and KI paper held over crucible will detect any nitric oxide given off ; (3) Cool and weigh ; (4) Difference from weight of dish gives fixed solids in amount of sample taken. Calcu- late as before. Volatile Solids. — Total solids less fixed solids, gives volatile. Consist of organic matter, nitrates, nitrites, ammoniacal salts, combined water, combined carbonic acid, and sometimes chlorides. Should not exceed 1-5 per 100,000 in a very good water. Example. — Evaporated 200 c.c. sample water to dryness, dried in air-oven, cooled in dessicator, and weighed : — Weight . . . . . . 19-674 grammes. Weight of platinum dish.. 19*554 ,, Difference . . . . . . 0-120 gramme. i.e., 0-120 grm. in 200 c.c. water, or 0-060 grm. in 100 c.c. or 100 grm., or 60 grm. in 100,000 grm., or 60 parts per 100,000 parts. Incinerated total residue at low heat. No blackening, fumes, odour, nor change in starch and KI paper. Cooled and weighed : — Weight . . . . . . 19-650 grammes. Weight of platinum dish . . 19-554 Difference . . . . . . 0-096 gramme. 24 PUBLIC HEALTH CHEMISTRY i.e. 0-096 grm. of fixed residue in 200 c.c. of sample water, or 0-048 grm. in 100 c.c. or in 100 grm., or 48 parts of fixed solids per 100,000 parts of sample. Then volatile solids = 60 — 48 = 12 parts per 100,000 parts. Chlorine (present as Chlorides). — This is estimated by precipitation with silver nitrate — the end of the process being known by the use of a few drops of potassium chromate, which gives a red precipitate with silver nitrate. So long as there is any chlorine in solution, however, the red precipitate which forms when each drop of silver nitrate solution is added, is immediately dispelled. The first indication of the red colour persisting is taken as the end of the reaction. The water sample must be neutral, and certainly not acid. It should also be colourless, or nearly so. Solutions required : (1) 5 per cent solution of potassium monochromate, K 2CrO 4, free from chlorine ; (2) Silver nitrate solution, either decinormal or standard, say 1 c.c. = 1 mgr. CI. Process. — (1) Take 100 c.c. sample in a clean porcelain basin ; (2) Add a few drops of chromate solution, which gives the liquid a yellow tinge ; (3) Fill burette with silver nitrate solution, and level ; (4) Run in the solution drop by drop, stirring the while ; (5) Stop when the least permanent red tint is got ; (6) Calculate amount present in parts per 100,000, and grains per gallon. After a preliminary trial, the end reaction can be more accurately watched, and a second estimation should always be done. Example. — 100 c.c. sample took 6-5 c.c. standard silver nitrate solution, of which 1 c.c. == 1 mgr. CI. Therefore, there are — 6-5 X 1 = 6-5 mgr. CI in 100 c.c. sample in 100 grm. ,, in 100,000 mgr. of sample that is, 6-5 parts per 100,000 parts. For grains per gallon multiply result by 0-7, thus : — 6-5 x 0-7 ass 4-55 grains per gallon. The result is sometimes required in terms of NaCl. WATER ANALYSIS 25 Every molecule of NaCl = 58-5 parts, of which 35-5 parts are CI. Therefore one part of CI === 58-5 -r- 35-5 = 1-65 part NaCl. Then 6*5 parts CI per 100,000 parts of sample becomes 6-5 X 1*65= 107 parts NaCl per 100,000 parts of sample. With decinormal silver nitrate solution the process is similar, but as 1 c.c. = 3-55 mgr. of CI, much less solu- tion will be required. On this account a larger quantity of sample is frequently taken, say 250 c.c. When more than 10 c.c. of standard silver solution are required in the titration, it is advisable to repeat the process after diluting the sample water with distilled water. In this way a more accurate result is obtained. The purest water as a rule contains less than 1-5 parts CI per 100,000. Increase may be due to sea-water, salt- bearing strata, sewage, etc., and gives cause for suspicion until explained satisfactorily. Poisonous Metals. — Under this heading are usually included Pb, Cu, Fe, and Zn. The presence of lead, copper, or iron in appreciable amount can be determined by taking 100 c.c. in a Nessler glass, and adding one or two drops of ammonium sulphide solution, when some darkening of the sample will occur in proportion to the quantity present. If no change is noted, compared with a control, then the sample will require to be concentrated, and tests applied. A delicate qualitative test is to take two 100 c.c. Nessler glasses, and fill one to the 100 c.c. mark with sample and the other with distilled water. To each then add a few drops of solution of permanganate of potash to give them a distinct pink tinge. Then add to each 1 c.c. of sulphuric acid and 1 c.c. of potassium ferrocyanide, and compare the tints. Iron gives a blue tint ; copper gives a brown ; zinc gives a white ; and lead gives no change. The control shows no change. Qualitative Test Table. — Concentrate sample to one- fifth of its bulk, say 200 c.c. to 40 c.c, and test thus : — To 5 c.c. in a test tube add a few drops of Am 2S solution. Black precipitate may be lead, copper, or iron. White ,, ,, zinc. 26 PUBLIC HEALTH CHEMISTRY i. If precipitate is black, divide into two portions. a. To one portion add dilute HC1 ; if precipitate dis- solves, then iron is present. Confirm for iron. Take two tubes with 5 c.c. of concentrated sample in each, after heating for a few minutes with a pinch of potassium chlorate to oxidize the iron to the ferric state. To one, add solution of KCNS ; blood-red colour produced. To other, add solution of K 4FeCy 6 ; prussian blue colour. b. To other portion add KCN ; if precipitate dissolves, copper present. Confirm for copper. As above, take two tubes with concentrated solution, but without treating in any way. To one, add AmOH ; blue colour. To other, add K4FeCy6 ; bronze precipitate. c. If precipitate does not dissolve ; then lead. Confirm for lead. As above, take two tubes with concentrated solution. To one add KI solution ; yellow precipitate, soluble on boiling. To other add K 2CrO 4 solution ; yellow precipitate, soluble in KOH. 2. If precipitate is white, confirm for zinc. Take two tubes as before. To one, add AmCl, AmOH and Am 2S ; white precipitate. To other, add K4FeCy6; white gelatinous precipitate. Arsenic. — Take a litre of the water sample, add pure sodium carbonate until alkaline, and evaporate nearly to dryness. Test concentrated liquid by Reinsch's or Marsh's test. The former is described under Beer (page 129). Tin. — Evaporate a litre of water sample to dryness, ash the residue; exhaust ash repeatedly with strong HC1, evapor- ate portions to dryness on water-bath, add some water, boil, and filter. Test filtrate with H 2S ; a dingy, yellow precipitate, soluble in Am2S, indicates the presence of tin. The precipitate is also soluble in the caustic alkalies. Quantitative Estimation. — This depends on a colori- metric method. 100 c.c. of the sample water are taken in a Nessler glass, and a measured quantity of a suitable precipi- tating agent is added. According to the amount of metal present, a certain depth of coloration is obtained. 100 c.c. of distilled water are now taken in a Nessler glass, and the same quantity of precipitant added to them. From a burette, successive small quantities of a standard solution of the. metal being tested for are added to the glass containing WATER ANALYSIS 27 the distilled water ; and after each addition the coloration produced is compared with that in the glass containing the sample water. This is done by putting both glasses together on a white tile, or on a Nessler stand, and looking down through the liquids. If the tints are alike in depth, then the glasses have been matched. If the sample is the darker, more standard will require to be added to the other glass, and a further comparison made. If the sample is the lighter, a fresh amount of distilled water will require to be put up, and less standard solution added at first. In any case the comparisons are continued until matching of the tints is obtained, and then the amount of standard solution which has been added to the distilled water is held to measure the amount of metal present in the ioo c.c. of sample water. The process of comparing the tints or colorations to a match is called " Nesslerizing," and will frequently be used in subsequent work. Determination of Lead. — A standard solution of lead acetate is required, of such a strength that i c.c. = o-i mgr. (TV mgr.) of lead. Pb (C2H302)2 + 3H20; the molecular weight = 379, and contains 207 parts of lead. Therefore 379/207 == 1-83 parts of lead acetate contain 1 part of lead. That is, 1-83 grm. of lead acetate contain 1 gram, of lead. If we dissolve 1-83 grm. of lead acetate in 1 litre of distilled water, 1 c.c. will contain 1 mgr. of lead. This solution diluted ten times gives a standard solution of lead acetate such that 1 c.c. = o-i mgr. Pb. A solution of Am2S in water is also required. Process. — Take two 100 c.c. Nessler glasses, with distinctive marks affixed. To one add 100 c.c. of sample water. To the other add 100 c.c. distilled water. To both add 2 c.c. of Am2S solution, and stir. Now take a burette, and fill it with the standard solution of lead acetate. Add 1 c.c. of this to the distilled water and stir. Compare coloration produced with that in glass containing sample. If the sample is darker, add another c.c. of standard solution to the other glass, stir, and compare. Repeat procedure until a match is obtained. If the sample is lighter than the coloration produced by 1 c.c. of standard solution, begin again and add -| c.c. of standard solution. 28 PUBLIC HEALTH CHEMISTRY Example. — Suppose 3 c.c. of standard solution were required to match sample. 1 c.c. of standard solution lead acetate = o-i mgr. Pb. Therefore 3 c.c. of standard solu- tion lead acetate = 0-3 mgr. Pb. Hence there is 0-3 mgr. Pb in 100 c.c. of sample water, or 0*3 mgr. Pb in 100 grm. of sample water, or 0-3 mgr. Pb in 100,000 mgr. of sample water ; that is, 0-3 part of Pb in 100,000 parts of sample water. By this method 0-05 part per 100,000, or JF gr. per gallon, may be easily detected. Many waters, especially soft and peaty waters, possess a coloration sufficient to equal that produced by 0-5 c.c., or even 1 c.c. of standard lead solution. In such a case, carefully match the natural coloration in terms of the standard solution, and deduct the amount obtained from the amount required in the regular test. Where the coloration is deeper still, and a poisonous metal is sus- pected, evaporate 100 c.c. to dryness, ignite to get rid of vegetable colouring matter, digest the ash with HC1, filter, collect filtrate, washing filter-paper with distilled water, and make up bulk of filtrate to 100 c.c. Now test as before. As lead is a cumulative poison, its presence in a water should disqualify that water for domestic use. Copper. — Copper is similarly estimated, using a standard solution of copper sulphate, CuSO 4 + 5H 20. The mole- cular weight of this salt in crystals is 249, and as this amount contains 63 parts of copper, 249/63 or 3-95 parts will yield 1 part of copper. Hence 3-95 grm. of copper sulphate crystals dissolved in 1 litre of distilled water gives a solution such that 1 c.c. contains 1 mgr. of copper. This diluted ten times gives the desired standard solution, 1 c.c. = o-i mgr. Cu. Copper can also be estimated by precipitation with HC1 and potassium ferrocyanide, which gives a bronze color^ ation. Iron. — Iron is best estimated by oxidizing it, if necessary, to the ferric state, and then adding potassium sulpho- cyanate, which produces a blood-red colour. Reagents required : (1) Standard solution of ferrous sulphate, FeSO 4 + 7H 20, 0-496 grm. dissolved in 1 litre of distilled water (acidified with H2S04), 1 c.c. of this solution = 0'i mgr. Fe. Dilute ten times in use, then 1 c.c. = WATER ANALYSIS 29 o-oi mgr. Fe ; (2) Dilute HNO 3 solution : make up 30 c.c. of pure concentrated acid to 100 ex. with distilled water ; (3) Potassium sulphocyanate solution : 15 grm. KCNS dissolved in 100 c.c. of distilled water. Process. — To each of two Nessler glasses add 5 c.c. of each solution (2) and (3). Add 1 c.c. of standard solution to one, and to the other add a measured quantity of sample, say 10 c.c, and note depth of tint produced, compared to standard. If the two are near each other, proceed jas before to match. If the sample is much too dark, use less ; if much too light, use more. Always make up bulk in each Nessler to be approximately equal to each other by adding distilled water, before finally matching. If more than 3 c.c. of standard solution are required, the tint gets too deep. Iron is perceptible to taste when present to the extent of I gr. per gallon, or 1 part in 350,000 parts of water. Zinc is usually determined gravimetrically. It may be done volumetrically with standard solution of potassium ferrocyanide, using copper sulphate as an indicator, the brown copper precipitate not being formed until all the Zn is precipitated. The standard solution is made 0-324 grm. of K4Fe(CN) 6 . 3H 20 per litre. Then 1 c.c. = o-i mgr. Zn. Lime and Magnesia. — These are often present together from strata. Lime. — Ammonium oxalate gives a turbidity with 9 parts per 100,000, and a white precipitate with anything over 20 parts per 100,000. Magnesia. — Precipitate any lime present with ammonium oxalate, filter, then add AmCl, AmOH, and sodium phos- phate. Crystals of so-called triple phosphate will separate out in twenty-four hours. (MgNH4P04.) For this and the next test it is better to concentrate to «V Phosphates. — Add some dilute nitric acid, stir, add excess of ammonium molybdate, and heat. If phosphates are present, a yellow colour will form. Sulphates. — Add dilute HC1 and barium chloride solution — a white precipitate of sulphate of barium,, insoluble in all acids. 30 PUBLIC HEALTH CHEMISTRY Quantitative Estimation of Lime, Magnesia, Phosphates, and Sulphates. These are all estimated for gravimetrically by precipita- ting as above, collecting the precipitate, which settles after twelve hours, on a filter-paper of known ash ; drying, and igniting ; and weighing ash. The ash of lime is weighed as calcium carbonate, of magnesia as magnesium pyro- phosphate (Mg2P207), of sulphates as barium sulphate. The phosphates are precipitated as triple phosphates of magnesium and weighed similarly. In the case of magne- sium, the lime salts must be removed before precipitating, hence the filtrate from the lime estimation is suitable for the purpose. GASES IN WATER. Carbon Dioxide in Water. — Exists as free CO 2 ; bicar- bonate and carbonate ; and free CO 2 and bicarbonate. i. Free C02. — Determined by titration with N/20 sodium carbonate solution, using phenolphthalein as indi- cator. On adding the N/20 solution from a burette to the sample, a red colour appears which gradually fades as the carbonate absorbs the free carbon dioxide and changes to bicarbonate, which is neutral to phenolphthalein. Na2C03 + C02 + H20 = 2NaHC03 106 44 N/i sod. carb. = 53 grm. per litre and absorbs 22 grm. carbon dioxide per litre ; hence N/20 sod. carb. = 2-65 grm. per litre and absorbs i-i grm. carbon dioxide per litre; hence 1 c.c. N/20 will absorb o-oon grm. free C02. Process. — Take 100 c.c. of sample in an Erlenmeyer flask on a white slab, and add one drop of 1 per cent alcoholic solution of phenolphthalein. Fill burette with N /20 solution of sodium carbonate and add to sample, c.c. by c.c, until a permanent red tint remains after waiting a minute or two. Calculate out result. Free carbon dioxide is almost constantly present in ground waters, and in inverse ratio to the amount of dissolved oxygen. It may be as high as 13 parts per WATER ANALYSIS 31 100,000, probably derived irom ground air, increasing with the depth and decreasing with the porosity of the soil. 2. Carbonate and Bicarbonate. — The carbonate is first determined by titration oi the sample with the standard solution of oxalic acid, in presence of phenolphthalein, which gives a pink colour with carbonates and alkalies, and is colourless with bicarbonates and acids. H 2C 20 42,H 20+2Na 2C0 3 =*= 2NaHC0 8+Na 2C 20 4+2H 20 90 + 36 2 x 106 From the equation we see that 126 grim of crystallized oxalic acid react with 212 grm. of sodium carbonate, containing 88 grm. of C02, and change it to sodium bicarbonate. Thus 126 grm. of oxalic acid crystals measure the change of 88 grm. of G02 from the state of carbonate to that of bicarbonate, or 126/88 = 1-43 grm. measure the change in 1 grm. of C02. Hence a standard solution of oxalic acid crystals, 1-43 grm. per litre, is of such a strength that 1 litre == 1 grm. CO 2, or 1 c.c. = imgr. C02. When all the carbonate is changed to bicarbonate, the solution becomes colourless, and the number of c.c. used measures the carbonate present. Boil the fluid in the flask briskly for ten minutes, when all the bicarbonate, whether originally present or derived from carbonate, is decom- posed with formation of carbonate (signalized by the return of the pink colour) and evolution of C02, as shown thus : — 2NaHC0 3 - Na 2C0 3 + H 20 + CO 2 Cool and repeat the titration with the standard oxalic solution ; the number of c.c. required measures the amount of carbonate now in the sample. But this figure represents only half the C02 present before boiling, halt of the CO 2 having gone into the atmosphere. Hence the figure has to be doubled, and then measures the total amount of C02 present originally in the sample. The amount present as carbonate is known from the first titration, and so the amount present as bicarbonate is measured by the difference between the number of c.c. used in the first titration, and double the number used in the second titration. The rationale may be shown thus : — 32 PUBLIC HEALTH CHEMISTRY (2), NaHC08+ j fi b u d=N CQ +R 0+co (3). 2Na 2C0 3+H 2C ,0 4+Phth=2NaHC0 3+Na 2C 20 4 Example : ioo c.c. of sample, plus phth., required 8 c.c. of std. oxalic sol. to decolorize. Boiled ; cooled ; and titrated again, when n c.c. were required. COo as Carbonate, 8 parts per 100,000. CO._, as Bicarbonate, 22 — 8 = 14 ,, ,, 3. Free Carbon Dioxide and Bicarbonate. — These are estimated by the addition of excess of alkali in the shape of a known quantity of baryta solution. The baryta fixes the free carbon dioxide and that half bound in the bicarbonates, precipitating both as barium carbonate. The excess of alkali which remains unused is measured by titration with standard oxalic acid solution. C02 + BaH202 a* BaC03 + H20 CaCO 3C0 2 + BaH 20 2 = BaCO 8 + CaCO , + 2H 20 H2C204 -f BaH202 = BaC204 + 2H26 From these equations we see that one molecule of oxalic acid and one molecule of C02 are each able to neutralize one molecule of baryta in solution. Therefore, 126 grm. of crystallized oxalic acid neutralizes 171 grm. of baryta, which fixes 44 grm. of C02; or 126/44 = 2-86 grm. of oxalic neutralizes 171/44 grm. of baryta, which fixes 44/44 = 1 grm. CO 2. Hence a standard solution of oxalic acid 2-86 grm. per litre is such that 1 c.c. measures the quantity of baryta which fixes 1 mgr. of CO 2. Process.— To 100 c.c. of sample in a flask or bottle, add a drop of phenolphthalein, and then 5 c.c. of BaCl2 solution, 5 c.c. of AmCl solution, and 40 c.c. of baryta solution (0*5 per cent). If excess of baryta is present, the liquid should become red, and remain so. Stopper the vessel and set aside for twelve hours. Thereafter titrate the whole sample (or an aliquot part) with standard oxalic acid solution. Titrate 40 c.c. of fresh baryta solution. The difference between the number of c.c. required for the fresh baryta solution and that required for the baryta mixed with sample, measured in c.c. of standard oxalic solution the amount of baryta used up in fixing free C02 and changing bicarbonate WATER ANALYSIS 3a to carbonate. But as each' c.c. of oxalic equals I mgr. of CO 2, then the number of them gives the total number of mgr. of CO 2 in the water sample. The free C02 is determined as before by N/20 sodium carbonate solution, and the difference gives the amount present as bicarbonate. Dissolved Oxygen. — Winkler, Dibdin, Thresh, and Mohr have all devised methods for determining the amount of oxygen dissolved in water samples. Any such method must be simple, speedy, and accurate, and the water must not be operated on in an inert atmosphere, or there might be a rapid loss by diffusion. Winkler's is perhaps the most simple and readily applied, and needs no special apparatus. The following solutions are required : (a) Manganous chloride solution free from iron (80 grm. MnCl 2 + 4-H 20 in 100 c.c. of distilled water ; (b) KI and NaOH solution (10 grm. KI in 100 c.c. of 33 per cent NaOH). This solution when diluted, and sulphuric and starch solution added, should not give any blue colour ; (c) N/100 iodine (1-27 grm. I and 2 grm. KI dissolved in 1 litre). This is used to standardize thiosulphate ; (d) N/100 thiosulphate of soda solution (2-48 grm. Na2S203 -f 5H20 per litre) ; (e) Starch solution. Process. — Take a glass bottle provided with a well- fitting glass stopper and of about 300 c.c. capacity. Determine accurately the capacity when stoppered. Wash it out with some of the water to be examined, and then fill it to overflowing with sample water (avoid splashing). Introduce, by different pipettes, 1 c.c. of each of solutions (a) and (b), doing this carefully so that they are delivered close to the bottom of the bottle. Put in the stopper tightly, enclosing no air bubbles. Mix the contents by lightly swinging the bottle. A precipitate forms which is allowed to settle. This takes a variable time; usually fifteen minutes is sufficient. When it has settled and the upper part of the fluid is clear, introduce by pipette, so as to fall on to the precipitate, 5 c.c. of strong HC1, replace stopper and swing until precipitate dissolves, when the fluid becomes yellow-coloured from liberated iodine. The contents of the bottle are now poured into a clean beaker, the bottle washed out with distilled water, and the wash- ings added. It is then titrated with the N /ioo thiosulphate 3 34 PUBLIC HEALTH CHEMISTRY solution, every c.c. of which used equals o- 00008 grm. O, or 0-055825 c.c. oxygen. Starch solution is used for the end reaction. The amount of oxygen found is present in the capacity of the bottle, less the 2 c.c. of solutions added. The result is returned in parts by weight per 100,000, or in cubic centimetres per litre. 1. 2MnCla + 4NaOH = 4NaCl -f 2Mn(0H) 2. 2. 2Mn(0H) 2 + O + H20 = 2Mn(0H) 3. 3. 2Mn(OH) 3 + 6HC1 = 2MnCl3 + 6H 20. 4. 2MnCl3 + 2KI = 2MnCl2 + 2KCI + I2. The process must be done rapidly. Nitrites liberate iodine and so vitiate the result, increasing it. Much organic matter interferes with the method, as it absorbs the liberated iodine, thus diminishing the result. Rapid working diminishes the latter interference. The amount of dissolved oxygen in a water is influenced mainly by temperature, being less in summer and more in winter. Ordinary tap water in this country contains on an average 7 c.c. per litre, which is about 1 part by weight in 100,000. Water is saturated at 50 C, io° C, 150 C, and 20° C. respectively, by 8-68 c.c, 777 c.c, 6-96 c.c, and 6-28 c.c per litre. Suphuretted Hydrogen. — This is estimated by titration with N /ioo iodine, which is decolorized by the H 2S ; thus H2S + I2 = 2HI + S. Process. — Take 10 c.c N/100 iodine in a white porcelain basin. Fill a burette with sample, and add to basin until colour is gone, using starch for end reaction. The N/100 iodine is made as above, and is standardized against N/100 thiosulphate solution. Every c.c. of N/100 I equals 1 c.c. of N/100 H 2S. But N/i H 2S is 17 grm. per litre, therefore 1 c.c. N/i = 0-017 grm-> and 1 c.c N/100 = 0-00017 grm. H2S, or 0-17 mgr. Hence the number of c.c of N/100I used x 0-17, gives the number of mgr. of H 2S in the amount of sample run in from burette to decolorize the iodine. HARDNESS (TOTAL, TEMPORARY, OR PERMANENT). Hardness is due to the presence in a water of metallic salts which form insoluble compounds with the fatty acids usually present in soap. A soap is the oleate, stearate, WATER ANALYSIS 35 or palmitate of sodium or potassium. Hard soap has soda for its base, and soft soap has potash for its base. These soaps are soluble in water and form a lather there- with on shaking. When soap is used with a water in which lime, magnesia, baryta, iron, alumina, or other such substances are present, oleates, etc., of these bases are formed, which being insoluble are precipitated, and no lather can be produced until an excess of soap is present. A certain amount of the hardness is removable by boiling, and this is called temporary hardness, and is chiefly due to the carbonates of lime and magnesia held in solution by carbonic acid gas, and by sulphates of these, with salts of silica, alumina, and iron when present. The permanent hardness, or what still remains in solution after boiling, consists mainly of some sulphates, chlorides, and nitrates of calcium and magnesium, with a little iron and alumina. Free carbonic acid gas in water also consumes soap, two equivalents uniting with one of soap as ordinarily estimated. Amount of hardness is expressed as grains per gallon (Clark's degrees), or parts per 100,000 (metrical degrees) in terms of calcium carbonate. In Germany the hardness is expressed as metrical degrees of CaO per 100,000. The total hardness of a water should not exceed 30 parts per 100,000, if for domestic purposes. Hard waters vary from 20 to 30 degrees on the metrical scale ; a soft water from 8 to 15 ; and a very soft water from 8 downwards. The greater the permanent hardness, the more objectionable is the water ; and of a good water it should not exceed 50 metrical, or 30 to 40 Clark. Determination of Hardness. — 1. By Standard Soap Solution Method. Dissolve 10 grm. of castile or soft soap in 1 litre of a mixture of equal parts of distilled water and methylated spirit. Standardize the solution so that 1 c.c. completely precipitates 1 mgr. of calcium carbonate or an equivalent salt. The CaC03 may be dissolved in the least possible quantity of HC1, then evaporated to dryness twice, to get rid of the HC1, and then the resulting CaCl2 dissolved in the proper amount of distilled water ; that is, 1 grm. of the carbonate is treated as above, and the resulting 36 PUBLIC HEALTH CHEMISTRY chloride dissolved in i litre of aq. dest., then I c.c. = I mgr. CaCOg. The soap solution is then tested with 50 c.c. of aq. dest. (recently boiled to get rid of CO 2), to determine how much of it is required to produce a perma- nent lather ; that is, a lather which remains as a uniform film J in. thick on the surface of the water five minutes after the bottle has been laid on its side. The soap solution is added from a burette, y1^ c.c. at a time. The 50 c.c. are contained in a stoppered bottle, 150 c.c. capacity, which is well shaken after each addition, then laid on its side, and the character of the lather noted. If it quickly disappears, then more soap solution is added, until the lather has the permanence described. The amount usually required by 50 c.c. of aq. dest. varies from 0-2 c.c. to 0-6 c.c, and should be determined not once for all, but at intervals, as it will vary with the strength of the soap solution, and this latter tends to deteriorate on keeping. The soap solution is now standardized against the standard CaC03 solution, 5 c.c. of which are added to 45 c.c. of recently boiled aq. dest., contained in a glass- stoppered bottle of about 150 c.c. capacity. The soap solution ,is now added from a burette, 1 c.c. at a time. Shake briskly after each addition. When the proper lather is formed, the shaking of the bottle produces a soft sound which is different from the hard sound at first emitted, and heard when the bottle is held near the ear. Say that 4 c.c. of standard soap solution were required to produce the permanent lather, and that 0-5 c.c. were necessary for 50 c.c. of aq. dest., then 4 — 0-5 = 3*5 c.c. have been used in precipitating the 5 mgr. of CaCOg. contained in the 5 c.c. of standard calcium solution. But we wish the standard soap solution to be 5 c.c. = 5 mgr. or 1 c.c. = 1 mgr. calcium carbonate. Hence it is too strong, and we must dilute it (with a mixture of equal parts of methylated spirit and aq. dest.) so as to make every 3*5 c.c. up to 5 c.c, or every 35 c.c. up to 50 c.c. The solution is now of standard strength, but requires to be re-standardized at intervals, as it is somewhat unstable. The standardizing can also be done against a standard solution of Ba(N03)2 which has a molecular weight of 261 compared to 100 for CaC03. Therefore if 2*61 grnu WATER ANALYSIS 37 of barium nitrate be dissolved in I litre of aq. dest. I c.c. = i mgr. Ba(N03) 2 equal to I c.c. = I mgr. CaC03. Total Hardness. — Take 50 c.c. of sample in bottle and test with standard soap solution as above until a permanent lather is obtained. Deduct 0-5 c.c. (say) necessary to produce lather, and double the answer gives the total hardness in metrical degrees. This multiplied by 07 gives it in grains per gallon, or Clark's degrees. If more than 8 to 9 c.c. be required, it is advisable to dilute 25 c.c. of the sample with 25 c.c. recently boiled aq. dest., and redetermine the hardness. Permanent or Fixed Hardness. — Take 100 c.c. of sample water and make up to 200 c.c. with aq. dest. Boil down to one-half its bulk, and a little more. Allow to cool to 6o° F. (15-5° C), and make up to 100 c.c. with aq. dest. Remove 50 c.c. and determine hardness as before. By boiling, all the free and half-bound CO 2 is driven off, and nearly all the CaC03 is precipitated. The CaS04 and the CaCl2 are not affected if the evaporation is not carried too far. Some of the MgCO 3 at first thrown down is redissolved as the water cools. Temporary or Removable Hardness. — This is the difference between the total and the fixed hardnesses. Notes. — The lime salts precipitate at once with the soap solution ; the magnesium salts* precipitate slowly ; hence it sometimes happens that all the Ca is precipitated and a lather formed which shortly disappears, and more soap solution is needed. The presence of magnesium salts is said to cause the lather to be brown in colour and to break very easily. More soap is required to produce a lather with a certain amount of magnesium, than with the equivalent amount of Ca, though this is ignored in practice. 2. By Hehner's Alkalimetry Method. Temporary. — Titrate 50 or 100 c.c. of sample with N/50 H2S04, using methyl-orange as indicator, until a permanent pink is got. The sulphuric acid decomposes the CaCO 3 with evolution of CO 2, and until all the carbonate is decomposed, no sulphuric is free to attack the methyl- orange. The number of c.c. of sulphuric gives the number 38 PUBLIC HEALTH CHEMISTRY of mgr. of calcium carbonate in the amount of sample taken, because I c.c. N/50 H2S04 = 1 c.c. N/50 CaC03. But the molecular weight of CaC03 is 10O and N/50 = 1 grm. per litre, hence 1 c.c. = 1 mgr. CaC03. Permanent. — To a fresh lot of sample add sufficient N/50 Na2C03 to precipitate as carbonate all the Ca and Mg present, noting carefully amount used. Evaporate mixture to dryness on water-bath. Dissolve soluble part of residue in 10 to 20 c.c. of aq. dest. and filter through small filter-paper. Wash out dish and filter-paper with a little more distilled water to ensure complete removal of all the Na2C03. Titrate nitrate with N/50 H2S04, using methyl-orange as indicator, until a permanent pink colour is obtained. The difference between the number of c.c. of N/50 Na2C03 used and the number of c.c. of N/50 H2S04 required to neutralize, is the number of c.c. of N/50 sodium carbonate used up in precipitating the Ca and Mg. Every c.c. of same equals 1 c.c. of N/50 CaC03 = 1 mgr. CaC03. Thus we arrive at the amount of permanent hardness in the quantity of sample taken. Total. — The sum of the temporary and permanent hardnesses, as determined above, gives the total hardness. ORGANIC MATTER IN WATER. This is derived from vegetable and animal pollution, and is estimated in a variety of ways. Frankland's Method.- — The water is evaporated to a residue, which is ignited in a hard combustion tube with cupric oxide ; the evolved gases are collected and measured, and the amount of carbon and nitrogen found in these returned as Organic C and Organic N. In a good water suitable for domestic use, the Organic C should not exceed 0-2 part per 100,000, and the Organic N should not exceed 0-02 part per 100,000. The ratio of Organic C to Organic N furnishes a valuable indication of the nature of the organic matter present in unoxidized waters. Thus, unoxidized peaty waters give a high ratio of from 8 to 12 to 20 or even more, the average being about 12, and such a ratio is held to indicate organic matter of vegetable rather than of animal origin. In unpolluted upland surface WATER ANALYSIS 39 water, the ratio varies from 6 to 12 ; in surface water from cultivated lands, from 4 to 10 ; in shallow wells, from 2 to 8 ; in deep wells, and springs, from 2 to 6 ; in sea water it averages about 17 ; and in sewage it varies from 1 to 3, averaging about 2. Table. Unoxidized peaty waters Unpolluted upland surface waters . . Surface water from cultivated land Shallow wells Deep wells, and springs Sea water Sewage Organic C. 8 to 20 ; avge. 12 6 to 12 4 to 10 2 to 8 2 to 6 Avge. 1-7 1 to 3 ; avge. 2 Organic N. In waters subjected to oxidation, the ratio tends to be reduced when the organic matter is mainly vegetable, and the reverse when it is animal. Loch Katrine water (average of five years) gave Organic C, 0-148 part per 100,000, and Organic N, 0-016 part per 100,000, and the ratio as 9-2. This method is for trained chemists only. Wanklyn, Chapman, and Hall's Method recognizes that organic matter tends to resolve itself into simpler substances, and chooses to estimate the amount of ammonia present, free in solution or as salts, as an index of the amount of organic matter so resolved. Further, the water is so treated subsequently that any organic matter remaining undecomposed has its nitrogen split off as ammonia ; this is measured and furnishes an index of the amount of such organic matter. The absolute amounts of these two ammonias (the first called " the free and saline," the second " the albuminoid ammonia "), and their relative amounts, give valuable evidence of the state of a water with regard to organic pollution. Forschammer Process, as modified by Tidy, is commonly called Tidy's Process, and consists in measuring the oxygen- consuming or absorbing power of a water, and inferring therefrom the amount of organic matter present. It has many limitations, but under proper conditions furnishes another item on which to found an estimate of a water. 40 PUBLIC HEALTH CHEMISTRY Kjeldahl's Process, in which the organic nitrogen is converted into ammonia and estimated by distillation along with the free and saline ammonia. This method is very useful in highly polluted waters and sewage effluents, where the estimation of the albuminoid ammonia is tedious and difficult. It is much used to determine the total nitrogen in food-stuffs, from which the total proteins is got by multiplying by a factor which for meat foods is 6-25, and varies for other foods. Free and Saline Ammonia. — By Wanklyn's Method. — Solutions required : (1) Nessler's reagent. This is a saturated solution of mercuric-potassic- iodide in ammonia-free distilled water, the whole being rendered strongly alkaline with caustic soda or potash. It may be made thus : Dissolve 35 grm. of potassium iodide in 200 c.c. of ammonia-free distilled water and 12 -5 grm. of corrosive sublimate in 300 c.c. of ammonia- free distilled water. Add the iodide solution to the sublimate one, when a yellow to scarlet precipitate is obtained, which re-dissolves in the excess of potassium iodide present. (Mercuric iodide is almost insoluble in water.) HgCl2 + 2KI = Hgl2 + 2KCI. Hgl2 +2KI = HgI2.2KL Now add carefully a cold saturated solution of corrosive sublimate, stirring all the time, until a slight red precipitate remains permanent. In this way excess of potassium iodide, above that required to keep the mercuric iodide in solution, is used up. 120 grm. of caustic soda in stick are now added to the mixture and allowed to dissolve and cool. If the red precipitate has disappeared, add again a little of the saturated solution of corrosive sublimate, until a slight permanent red precipitate appears. Make up bulk to 1 litre with ammonia-free distilled water. The solution is now ready for use. Nessler's solution gives a yellowish tinge with the faintest trace of ammonia, and if much ammonia is present a yellow-brown precipitate forms of di-mercuric-ammonium- iodide : — NH3 + 2HgI2 = NHg2I + 3HL WATER ANALYSIS 41 (2) Standard solution of ammonium chloride, such that 1 c.c. = o-oi mgr. NH3. Ammonium chloride, NH4C1, has a molecular weight of 53-5, of which 17 parts are due to ammonia NH 3. Since the standard solution is 1 c.c. = o-oi mgr. of ammonia, 1 litre will contain o-oi grm. of ammonia. 17 : o-oi : : 53-5 : x = 0-03147 grm. of NH4C1 will yield o-oi grm. of ammonia. Hence, dissolve 0-03147 grm. of ammonium chloride in 1 litre of ammonia-free distilled water, and 1 c.c. will contain o-oi mgr. of ammonia. Process. — Take a retort or boiling-flask of about 700 c.c. capacity, cleanse it well and rinse it out with ammonia- free distilled water. Now put into it 200 c.c. of ammonia- free distilled water, connect to a condenser, start the water flow in latter, and distil over 100 c.c. to rid the apparatus of any traces of ammonia. Test the distillate with Nessler's solution, and if ammonia is found in the last portions, repeat the distillation. If not, cool flask, wash out with ammonia-free water, and proceed. Introduce into flask 500 c.c. of sample water and render this alkaline by the addition of some recently-heated sodium carbonate. Connect to a condenser, start the water supply for the latter, and place a clean 50 c.c. Nessler glass at the end of the condenser to catch the distillate. Make sure that all parts of the apparatus are properly connected and adjusted. Now apply the flame of a Bunsen burner to the flask, which may be protected by a piece of gauze. Heat gently at first, but once the parts have got heated, increase the flame. Try to distil over at the rate of 50 c.c. every fifteen minutes. When the first Nessler glass is filled to the 50 c.c. mark, remove it and put another clean one in its place, and so on. Have a stock of six ready for the purpose. The first 50 c.c. of distillate is then tested by adding to it 2 c.c. of Nessler solution and mixing. Place the glass on a white slab, or on the glass shelf of a Nessler stand, and on looking down through the liquid, the amount of coloration produced, or its absence, is easily made out. With experience the depth of colour will suggest how much standard solution will be required to match it. The next step is to put up three trial glasses for comparison. 42 PUBLIC HEALTH CHEMISTRY Take three 50 c.c. Nessler glasses, and from a burette add to the first 1 c.c., to the second 2 c.c, and to the third 3 c.c. of standard solution of ammonium chloride ; 1 c.c. = o-oi mgr. NH 3. Fill all three up to the 50 c.c. mark with ammonia-free distilled water, and then add to each 2 c.c. of Nessler solution, and mix. Put these glasses, distinctively marked, on the slab or shelf, and compare the first 50 c.c. of distillate with them as to depth of coloration. If the distillate matches any one of them, the result is attained. If it does not match any of them, it may be intermediate between any two of them, or be darker than the 3 c.c. or lighter than the 1 c.c. glass. In any case, fresh trial glasses should be put up for, as. the case may be, -5 c.c, 1-5 c.c, 2-5 c.c, 4 c.c, 5 c.c, 6 c.c, of standard solution. If the distillate is darker than the coloration given by 6 c.c. of standard solution, it is better to dilute it with ammonia-free distilled water, and then to proceed to match. The same procedure is carried out with the second 50 c.c. of distillate and succeeding lots. The distillation is stopped when no coloration is given with 2 c.c of Nessler, or at least less than will match 0-5 c.c of standard solution. This usually happens with the fourth lot of 50 c.c. The sum of the amounts of ammonia found in each lot of distillate, is the total free and saline ammonia present in 500 c.c. of sample water. This is reduced to the amount in 100 c.c, and thereafter expressed as parts of ammonia per 100,000. The distilling over in separate lots is the mode recommended by the Society of Public Analysts, but Wanklyn recommends that only 50 c.c. be distilled, and that the amount found in it be increased by one-third, on the ground that in his experience three-fourths of the ammonia comes over in the first lot of 50 c.c. Example. — First 50 c.c. of dist. matched 5.0 c.c. std. sol. of NH4C1. Second 50 c.c do. do. 1-5 c.c. do. do. Third 50 c.c. do. do. 0-5 c.c do. do. Fourth 50 c.c. do. do. nil 7-0 c.c do. do. WATER ANALYSIS 43 That is, the 500 c.c. of sample water yielded ammonia sufficient to match 7 c.c. of standard solution of ammo- nium chloride. (1 c.c. = o-oi mgr. NH3), hence 7 c.c. standard solution equals 7 X o-oi — 0-07 mgr. NH 3, and there is — 0-07 mgr. of free and saline NH3 in 500 c.c. of sample. or 0-014 mgr. of do. do. do. in 100 c.c. or do. in 100 grm. or do. in 100,000 mgr. or 0-014 Part of do. do. do. in 100,000 parts. Note. — In Nesslerizing, always add the AmCl solution first to the trial glasses, and the Nessler later. If the order is reversed, a turbidity is produced which prevents accurate comparisons. Likewise always use distilled water, ammonia-free. Pure natural water, ammonia-free, will not do, as it appears muddy when compared with distilled water. The residue in the flask is used to determine the " albuminoid ammonia," as now described. Albuminoid Ammonia — is determined by breaking up the nitrogenous organic matter in the water sample by the action of an alkaline solution of potassium permanganate, the nitrogen being converted into ammonia, which is distilled off and estimated as described for free and saline ammonia. All the nitrogenous organic matter is not so decomposed, but the proportion of it which does so is sufficiently uniform to form a basis for deductions. The albuminoid ammonia is approximately ' one-tenth of the nitrogenous organic matter in the water. Solution required is " alkaline permanganate," made by dissolving 8 grm. of potassium permanganate and 200 grm. of caustic potash in 1100 c.c. of distilled water, and boiling down the bulk to 1000 c.c. (1 litre). The amount of alkaline permanganate used should be about one-tenth of the bulk of sample taken. It should be mixed with three volumes of water, and boiled down to three volumes. That is, in present case, take 50 c.c. of alkaline permanganate, add to them 150 c.c. of water, and boil down to 150 c.c, which are added to the residue in 44 PUBLIC HEALTH CHEMISTRY flask. This gets rid of any free or saline or albuminoid ammonia in this added fluid. Process. — Take the residue from the estimation of free and saline ammonia, and keeping it still in the flask, add to it 50 c.c. of freshly-boiled permanganate solution, and about 100 c.c. of ammonia-free distilled water, to increase the bulk. Some fragments of pumice stone, which have been heated to redness in a Bunsen flame and cooled, are also added. The apparatus is now fixed together and distillation resumed, the distillate being collected as before in 50 c.c. Nessler glasses. The determination of the amount of ammonia is made in precisely the same way as for free and saline ammonia. The number of lots of 50 c.c. to be collected cannot be approximately stated, as the splitting up of the organic matter occurs irregularly, and in this way more ammonia may be found in the second or third lot than in the first. The process should be continued until no reaction with Nessler is got. In some cases it may be necessary to stop the process and allow the apparatus to cool, then add more distilled water, and then resume the distillation. The amount of ammonia found is of course derived from the original 500 c.c, and must be calculated accordingly. Free and saline ammonia represents the ammonia combined with carbonic, nitric, or other acids, and also what may be derived from urea, or other easily decom- posable substances, if present. The limit in pure waters is 0-002 mgr. per 100 c.c, and in a usable water it should not exceed 0*005 nigr. per 100 c.c. Albuminoid ammonia in drinking waters of good quality should not exceed o-oi parts per 100,000. Much albuminoid ammonia with a small amount of free ammonia usually indicates vegetable contamination, particularly if the chlorides and nitrates are low. Peaty waters yield large quantities of albuminoid ammonia, which is slowly evolved ; whereas badly polluted waters as a rule yield their high proportion more rapidly. Oxygen Absorption or Consuming Power. — Tidy's process is based on the fact that much of the organic matter in a water is capable of oxidation, and especially by permanganate in acid solution. Unfortunately, different WATER ANALYSIS 45 substances reduce different proportions of permanganate, and slight variations in temperature and acidity influence the readiness of the permanganate to part with its oxygen to an appreciable extent. Nevertheless, the process yields results which, taken in conjunction with other analytical facts, aid materially in forming an opinion of a water sample. Reagents required : (a) Standard solution of potassium permanganate : — 2K 2Mn 20 8+6H 2SO 4=2K 2SO 4-f 4MnSO 4+6H 20+50 2 2 x 316 5 x 32 ; that is, 632 parts of potassium permanganate liberate 160 parts of oxygen, or 1 part of O will be set free by 3-95 parts of permanganate. Hence, if 3-95 grm. of the latter be dissolved in 1 litre of aq. dest., then 1 c.c. = 1 mgr. O. The solution is usually diluted ten times in use, so that 10 c.c. =* 1 mgr. O. (b) KI solution, 10 per cent in aq. dest. (c) Starch solution, 1 grm. per half litre, freshly boiled and filtered, (d) Sodium thiosulphate solution, 1 grm. to the litre of distilled water, (e) Sulphuric acid, 25 per cent in aq. dest. Process. — Take two stoppered flasks or bottles of at least 300 c.c. capacity, and into one put 250 c.c. of sample water, and into the other put 250 c.c. of distilled water. To each add 10 c.c. of the 25 per cent sulphuric acid, and place them both on a water-bath at 8o° F. or 260 C. When the required temperature is reached, 10 c.c. of the perman- ganate solution are added to each lot. A pink colour will result. Maintain the temperature, and observe carefully whether the pink colour is discharged. If so, then another 10 c.c. of the permanganate solution is added to the sample and the control, and more if necessary to keep them markedly pink. Further addition of sulphuric acid is not needed. At the end of a specified time, which may be fifteen minutes, half an hour, one hour, two hours, three hours, or four hours, or any combination of these (the commonest being fifteen minutes and four hours), the oxidizing process is stopped by the addition of 1 c.c. of the KI solution, when the unused permanganate reacts thus,, through its loosely held oxygen ; 5O 2 + 20KI + 10H 20 =. 46 PUBLIC HEALTH CHEMISTRY 20KOH + 10I 2. The liquid turns a yellow colour from the iodine set free. The quantity of iodine liberated is strictly proportional to the amount of unused perman- ganate. It remains, therefore, to estimate the amount of iodine set free, which measures the amount of oxygen unused, and this deducted from the amount known to have been added, gives the amount absorbed. This is done by titrating the yellow solution with the thiosulphate solution until the yellow colour is nearly gone, and then adding 1 c.c. of starch solution to give a more distinct end reaction. The titration is finished when the blue is just gone : — 1 2 + 2Na 2S 20 3 = 2NaI -f- Na 2S 40 6. Both the sample and the control are treated thus. In the control presumably no oxidation takes place, so that the number of c.c. of thiosulphate solution required for it, is a measure of the iodine liberated by all the oxygen free to cause oxidation. The amount of thiosulphate solution used for the sample measures the unused oxygen, and the differ- ence between these two numbers gives the proportion of oxygen used up. For example, say that 10 c.c. only of per- manganate were required to be used, and that after adding KI solution the control took 40 c.c. of thiosulphate solution to decolorize, and the sample took 30 c.c. ; then 40 c.c. of thiosulphate measure 1 mgr. of oxygen, and 40 c.c. — 30 c.c. = 10 c.c. measure 10/40 = 0-25 mgr. O, and this is the quantity absorbed by the 250 c.c. of sample taken. This multiplied by 0-4 gives o-i mgr. O absorbed per 100 c.c, or o-i part per 100,000. Waters of great organic purity will not consume more than 0-05 part of oxygen per 100,000 in fifteen minutes at 8o° F., and if the amount absorbed exceeds o-i part in fifteen minutes, the sample may be considered of doubtful purity. After four hours' exposure, an absorption of more than 0-3 part must be regarded with suspicion. Ferrous salts, nitrites, and sulphuretted hydrogen, if present, vitiate the test. Kjeldahl's Process for the determination of organic nitrogen is performed only in very polluted waters. The process is described under Sewage and Sewage Effluents. WATER ANALYSIS 47 NITRITES AND NITRATES IN WATER. Ammonia present in water, derived either from the decomposition of organic matter or by synthesis from urea, tends in its passage through the soil to become oxidized, first into nitrites, then into nitrates. Nitrates, however, may be present in water which has dissolved it out of strata through which it has passed. Sometimes these nitrates become reduced, first to nitrites, then to ammonia, and this has been specially observed as due to iron salts in the ferrous state. In the London basin, the deep-well waters from the " greensands " strata have been noted as yielding ammonia thus derived. Waters polluted with vegetable matter yield little nitrites and nitrates relatively, as plant life removes these, and vegetable matter contains little nitrogen. Nitrites. — Qualitative Tests. i. Starch Iodine Test. — Take 50 c.c. of sample water in a Nessler glass and 50 c.c. distilled water in another. To each add a few drops of KI solution and a few drops of freshly-made starch solution. Now add a few drops of dilute sulphuric acid to each tube. The presence of nitrites is indicated by an immediate blue colour. 2. Naphthylamine Test. — Take two Nessler glasses as above and acidulate with acetic acid. Add to each a few drops of naphthylamine solution in sulphanilic acid. With nitrites a beautiful pink colour develops in two to three minutes. Quantitative Tests. Griess's Test. — Solutions required : — a. Metaphenylene-diamine solution, 5 grm. per litre, slightly acidulated with sulphuric acid, decolorized by boiling with pure animal charcoal. b. Sulphuric acid, one part of strong acid to two parts of aq. dest. c. Standard nitrite solution. This is made from silver nitrite because it is the most stable salt. AgNO 2+KCl = AgCl + KNO 2. 154 parts of silver nitrite, when treated with KG, give rise to 85 parts of KNO 2, or 46 parts of nitrous acid as represented by N02, or 308 parts are equivalent to 76 parts of N 20 3. Hence, if 308 /y6 = 4-06 48 PUBLIC HEALTH CHEMISTRY grm. silver nitrite are dissolved in boiling distilled water, and precipitated by slight excess of KC1, I grm. of nitrous acid as N203 is left in solution in combination with potassium. The bulk is made up to I litre, the precipitate allowed to settle, and 10 c.c. are taken and diluted to I litre, then i c.c. = o-oi mgr. N203 (equal to i per 100,000). Standard nitrite solution is also made so that 1 c.c. = o-oi mgr. N, and also of millinormal strength, and then 1 c.c. N/1000 = 0-046 mgr. N02. Process. — To 100 c.c. of sample in a Nessler glass, 1 c.c. of the dilute sulphuric acid and 1 c.c. of the metaphenylene- diamine solution are added as a preliminary test. If an orange colour is immediately produced, the tint will prove too deep for comparison. In such a case 50 c.c. should be tried, and if found suitable such an amount diluted to 100 c.c. with aq. dest. is to be used in the real test, which is done thus : — Having decided the amount of the sample to be used, it is taken in a 100 c.c. Nessler glass and made up to 100 c.c. if required. Three other Nessler glasses are taken, and 1 c.c, 2 c.c, and 3 c.c. of standard nitrite solution added to each respectively, and the bulk is made up to 100 c.c in each case with aq. dest. To all the glasses is added 1 c.c. of each of the reagents, namely M-P-D and H2S04, and this is done as quickly as possible, so that the colours in the glasses may develop from as nearly as possible the same time. The glasses are set aside for fifteen to twenty minutes and are then compared in the manner known as " Nesslerizing." If the sample matches one of the standards, then the amount in it is known. If not, fresh trial glasses are put up, the amount of standard being gauged from the preceding experiment. The colour produced is Bismarck brown or triamido-azo- benzol. Griess's test is a very accurate one but requires that the water and the reagent should be colourless or be decolor- ized. The reagent may be bleached by pure animal charcoal. Ilosvay's N aphthylamine Test. — a. Solution of sulphanilic acid 0-5 grm. in 150 c.c of diluted acetic acid (specific gravity 1-04). b. Solution of naphthylamine made by dissolving o-i grm. in 20 c.c of aq. dest., filtering, and adding 180 c.c diluted acetic WATER ANALYSIS 49 Process. — Take ioo c.c. of sample in a Nessler glass, and in another the same quantity of aq. dest. and I c.c. standard nitrite solution. To each add 2 c.c. of each of the above solutions, a and b. Set aside for five minutes and then compare tints. If not equal in tint, abstract some fluid from the darker by pipette and make up the bulk with aq. dest. If the colours still do not match, more fluid is removed, and bulk made up as before. Suppose sample is darker, and that 40 c.c. are removed, and bulk made up ; and that again, 30 c.c. are removed, when finally tints match. Then we get : — 100 x 60/100 x 70/100 = 42 c.c. of the original 100 c.c. match 1 c.c. of standard nitrite solution, say 1 c.c. = o-oi mgr. N : then 42 c.c. of sample contain o-oi mgr. N, and therefore 100 c.c. will contain o-oi x 100 ~- 42 = 0-023 mgr- N, or 0-023 Part of N as nitrite per 100,000 parts. The nitrites first act on the sulphanilic acid and form a new compound which reacts with the naphthylamine and forms the substance which gives the pink colour to the liquid. A water containing nitrites is not safe for domestic use, and should be rejected on that evidence alone, unless unexceptionable in all other respects. Nitrates. — Qualitative Tests. 1. Brucine Test. — Take 5 c.c. of sample and add 5 c.c. of brucine solution (1 in 1000), then mix, and pour carefully down the side of the test tube some pure, strong sulphuric acid, free from nitrates, when a positive result is denoted by the appearance of a pink ring at the junction of the liquids on gentle shaking. The test is also performed by evaporating 10 c.c. of sample to dryness in a clean porcelain basin, then adding a crystal of brucine, and then allowing one drop of pure sulphuric to run down the side of the dish, over the solids ; when in the presence of nitrates a pink is obtained. Detects 0-7 part per 100,000. Unreliable in the presence of nitrites, which should be first destroyed by addition of urea and sulphuric acid to sample ; allow to stand aside for an hour, when test can be applied as before. 4 50 PUBLIC HEALTH CHEMISTRY 2. Diphenylamine Test (C6H5)2NH. — Take 5 c.c. of sample, add as much diphenylamine solution, mix, and run down pure strong sulphuric, when a blue colour forms at junction of liquids in presence of nitrates. Quantitative Tests. 1. Phenol-sulphonic Acid. — This reagent is made by adding 6 grm. of pure carbolic acid to 3 c.c. of aq. dest. and then adding mixture to 37 c.c. of pure sulphuric acid. A standard solution of potassium nitrate is required, 0-072 grm. to 1 litre, and then 1 c.c. = o-oi mgr. N as nitrates. This contains 1 part N in 100,000 parts of standard. Process. — To two porcelain dishes are added respectively 10 c.c. of the sample and 10 c.c. of the standard. These are placed on the water-bath until their contents are just evaporated to dryness. To each of the residues add 1 c.c. of phenol-sulphonic acid, and mix well with a glass rod (if a large amount of nitrates is present the liquid will turn red). Set aside for fifteen minutes, and then wash out each dish successively into two clean 100 c.c. Nessler glasses with 25 per cent ammonia solution in distilled water. Add more ammonia until effervescence ceases, and make up to mark with aq. dest. The nitrates present convert the phenol-sulphonic acid into picric acid, with which the ammonia forms a picrate having a yellow colour, and the amount of this is proportional to the amount of nitrates present. The two glasses are now compared as to tints, and the darker one is diluted as before described under Ilosvay's test for nitrites. If the water is very pure, a larger amount of sample should be evaporated down, say 20 c.c, 50 c.c, or 100 c.c, and a smaller quantity of standard, say 5 c.c. If rich in nitrates, then less should be taken of the sample, say 5 c.c or 1 c.c Aluminium Process. — If aluminium foil be added to a strongly alkaline water, decomposition of the water ensues with the evolution of hydrogen, which in the presence of nitrites or nitrates reduces these, converting their contained nitrogen into ammonia. Thus : — 4AI + 4NaOH + 4H20 = 2Al2Na204 + 6H2 : and 3KN03 + I2H2 = 3KOH + 6H20 + 3NH3. WATER ANALYSIS 51 Required : (i) Thin aluminium foil ; (2) 10 per cent solution NaOH. Process. — Take 100 c.c. of the water sample and 100 c.c. of the NaOH solution in a 300 c.c. boiling-flask. Add a piece of aluminium foil about 1-5 inch square. Cover but do not cork. Set aside for six hours at least. Then connect the flask to a condenser and distil over the ammonia into 50 c.c. Nessler glasses, collecting three lots. The amount of ammonia is determined by comparison of the coloration developed in these glasses by adding to each 2 c.c. Nessler's solution, and that developed in glasses containing 50 c.c. ammonia- free distilled water, plus 1 c.c, 2 c.c, and 3 c.c respectively of standard NH4C1 (1 c.c. = o-oi mgr. NH3), and similarly treated. The amount estimated by this method includes ammonia present in the sample, ammonia derived from nitrites, and ammonia derived from nitrates. The two former are separately estimated and deducted, and the remainder is the amount derived from nitrates, and is readily converted back into terms of NO 3 or of N. Example. — 100 c.c gave ammonia equal to 40 c.c of standard AmCl = 40 x o-oi = 0-4 mgr. NH3. But the water contained 0-006 mgr. of free and saline ammonia per 100 c.c, and 0-042 mgr. N as nitrites per 100 c.c = 0-042 x 17 -5- 14 = 0'05iNH3 per 100 c.c. Hence, 0-4 — (0-006 4- 0*051) = 0-343 mgr. NH 3 due to nitrates = 0-282 mgr. N as nitrates per 100 c.c. or 100,000 mgr. Copper-Zinc Couple Method. — This method is similar in principle to the above. A bright piece of thin zinc foil, 3 in. X 2 in., is cleansed with dilute sulphuric acid. It is then rolled into a coil, so that it may fit into a 200 c.c. wide-mouthed bottle. Now immerse the coil for three minutes in a 3 per cent solution of copper sulphate. The zinc becomes coated with a black deposit of metallic copper. Remove the coil carefully, wash in ammonia-free distilled water, wash in sample water, and then immerse in no c.c of sample water contained in a wide-mouthed bottle. Stopper tightly, place in a cool dark place for twenty-four hours. The " Copper-zinc couple " acts electrically on the sample, changing any nitrates present 52 PUBLIC HEALTH CHEMISTRY to nitrites, and then to ammonia. It thus acts also on any nitrites originally present, so that the process estimates nitrates and nitrites. The reaction is finished when no free nitrites are found in the solution. This is determined by removing 10 c.c. and testing by Griess's test. If nitrites are found to be present, more time must be given. If they are absent, the remainder of the sample water is poured into a 700 c.c. boiling-flask, and the bottle washed out repeatedly with ammonia-free distilled water, the washings being added to the flask, and more water added to bring up the bulk to about 500 c.c. The water is then distilled as in the estimation of free and saline ammonia, and the amount of ammonia determined. This is restated as nitrogen by multiplying by 14/17 (N:NH3). If the sample was found to contain any free ammonia, the amount of this would require to be deducted before assigning the amount found by this process to nitrates and nitrites. Indigo Method. — Another method, which is a rapid and convenient one, but subject to great irregularity, is the indigo method. 20 c.c. of sample are taken in a beaker, and 20 c.c. of pure strong sulphuric acid are added. From a burette allow standard indigo solution to run into the hot mixture, until the colour of the indigo ceases to be discharged, and a faint greenish tinge becomes permanent. The estimation should be repeated, adding half a c.c. of indigo less to 20 c.c. of the sample, and then the sulphuric. When the colour is discharged, the indigo is run in drop by drop until colour is again permanent. The indigo solution is standardized against standard nitrate solution similarly treated. The strong sulphuric liberates free nitric acid, which in the hot liquid oxidizes the indigo to isatin, which is colourless. Owing to the heat evolved, the titration is best done with the beaker resting on an asbestos mat. The method is unreliable in the presence of organic matter, the results being too small. It also requires that all the procedures should be carried out exactly alike for the titration of the sample and the standardizing of the indigo solution. Otherwise it is a very simple, rapid, and delicate method. No water used for drinking purposes should contain WATER ANALYSIS 53 more than 0-35 part of nitrogen as nitrates per 100,000 parts, unless there is some satisfactory explanation. This amount equals about one grain per gallon when expressed as N205, or 1-5 parts per 100,000 when expressed as N03. ICE. Ice is frozen water, and it is not usually purer in content than the water from which it is derived. What- ever may be frozen out of the water is usually mineral matter, such as salt ; suspended matter is likely to be enclosed. Microbic content, when composed of the com- mon sewage organisms, is little affected by the temperature of freezing, for the most part only being rendered torpid. As far as possible, therefore, ice should only be used when made from pure water, and by a process in which it is not subject to risk of serious contamination. The analysis of ice proceeds on the same methods as for water, the ice being first melted. MINERAL WATERS AND AERATED WATERS. These are examined on the same principles. In the case of artificial waters, the spring or supply from which they are made should also be examined. In such also a search should be made for poisonous metals, such as lead and antimony, iron, copper, zinc, and even arsenic. In natural mineral waters the same careful examination should be made. In these the mineral content is often considerable, and a thorough analysis of the different metals present is very important. The temperature and the amount of carbonic acid gas are also noted. Nowadays the presence of metals of the radium group has acquired a new significance, and their occurrence is specially noted. INTERPRETATION OF THE RESULTS OF A WATER ANALYSIS. This must not be based on any one item, but on a careful consideration of the following points : 1. Local inspection for any source of possible pollution. 2. Bacteriological examination made as soon after collec- tion as possible. 54 PUBLIC HEALTH CHEMISTRY 3. Chemical analysis made as soon after collection as possible. It is rare for a sample of water to yield results under the second and third headings, where careful local inspection has failed to suggest danger of pollution. It should, there- fore, be thoroughly carried out. Bacteriological examination absolutely condemns a water for domestic use when pathogenic organisms are found in it. Unfortunately the detection of these is not always an easy matter, and so their presence or absence is inferred from the abundance or scarcity of associated forms which are more readily found and identified. The result of this is that the bacteriological examination mostly furnishes evidence confirmatory to that derived from other sources. Chemical analysis is more rapidly accomplished than the other procedures, and was formerly regarded as a sufficient basis for diagnosis of a water sample, in regard to its wholesomeness or otherwise for domestic purposes. This is so no longer, because it is recognized that the consti- tuents sought for and actually found in a particular water sample, for the most part are of themselves non-deleterious, and by their excess or deficiency simply suggest the pre- sence or absence of the actual materies morbi. Chemical evidence, therefore, must be used in conjunction with all the other evidence before a definite opinion is formed, and even then the judgment may be wholly based on negative findings, which here, as elsewhere, may at any time not bear the interpretation put upon them. Nevertheless, the following statements, when cautiously used, are helpful in interpreting results. High chlorine and oxidized nitrogen, associated with marked free and albuminoid ammonia, suggest present or recent animal pollution. High chlorine and oxidized nitrogen (not from strata), with little free and albuminoid ammonia, suggest past or remote animal pollution. Low chlorine and oxidized nitrogen, and very low free and saline ammonia with high albuminoid ammonia, suggest pollution of vegetable origin. Deep wells often show a large amount of chlorides and WATER ANALYSIS 55 free ammonia, without these necessarily indicating pollution. This is especially notable in wells sunk into strata like the London greensands. The organic nitrogen in a water is mostly determined by the estimation of the albuminoid ammonia which only partially measures it. If the organic nitrogen by Frank- land's process be I part per 100,000, then the albuminoid ammonia of the same water would be about 0*615 Part Per 100,000, containing 0*506 of organic nitrogen ; and the organic nitrogen by the Kjeldahl process would be about double that in the albuminoid ammonia, or 1-012 part per 100,000. Specimen An alyses (from Notter and Firth) Chlor- ine Free NH8 All). NH3 Oxygen absorbed Nitrates Nitrites 1. Upland surface ... 10 0 003 0012 0 290 016 nil 2. Shallow well 2*2 0011 0 009 0 200 0 002 nil 3. 10 nil 0 003 0 040 0 800 nil 4. 125 0005 0 006 0150 1500 traces 5. Deep well 2'8 0010 0 004 0 060 0030 nil 6. „ „ 290 0055 0 002 0110 0110 nil 7. M „ 190 0018 0 004 0110 0390 traces 8. ,, 220 0011 0 004 0 060 0 090 nil 9. Spring in a copse... 1*6 0020 0001 0015 rioo nil 10. ,, near ditch 40 nil 0 006 0 200 1700 nil 11. ,, in meadow 3'9 0008 0030 0180 0 200 nil 12. ,, protected... 30 0 009 0 006 0122 0 600 nil Notes. Local Inspection : In numbers 1, 2, 5, 8, and 12, sources of pollution were absent or well guarded against. Numbers 3 and 7 were in farmyards. In numbers 4, 6, 9, 10, and 11, defects in construction or in protection from possible pollution were found. Bacteriological Examination : In numbers 4, 6, 7, 10, and 11, sewage organisms were found. Opinion : Numbers 1, 3, 5, 8, and 12 were returned as safe; number 2 as doubtful; and numbers 4, 6, 7, 9, 10, and 11, as unsafe. 56 PUBLIC HEALTH CHEMISTRY SEWAGE AND SEWAGE EFFLUENTS. The subjoined table will suggest ideas on this subject. M 2 C5 c«s 3 I I I I 5 o I l 3 a m p i i i ^O 00 M O O I I O O M O ON o w C H 00 N O CO CO 1 O O | n, t>9 rfl o* CO m CO M N. »o 10 III a V en t»» 6 r^ fo ro 1 o o M M 1 hO G c u IN O M H Ml it Be re | O-+iO'0 1 -*■ m On On "ON 'tf) O I m On O O £ On O CO o ,0 III 6 i>. rr~.ch u-> 1 M M 1 no 66 h iO M o< 6 ill — ' — . en u-> N. N 9\"ti ^ f5 ^ V m 6 H 6 t^ >0 N NN t-* N re ."2 O * 2* " :n M IN Onm !>. t}- > G III c 'a ■) c CJO o a s a bo a O fl;fl ass fl S^ o :'fl O a3 flj +> QJ fl rt 5 H a.-s c3 PI . |a .S en co fl o +> g'C'fi ,Q +> +» fl •• 'aT""' . la <^ O u O fl OOO S fl £ •fl l886» Klebs-Loeffler 1883-84, Micrococcus melitensis 1887, B. enteritidis 1888, B. tetanus 1884-89, B. pestis 1894, \B. enteritidis sporogenes 1895, Cholera v. 1883, B. botulinus, B. paracolon and paratyphosus, and B. Morax-Axenfeld, all in 1896. CHAPTER IX. GENERAL PRINCIPLES. BACTERIOLOGICAL MEDIA may be thus classified : — Nutrient Broth, standardized. Derivatives : Glucose broth, lactose broth, nutrient gelatin, nutrient agar-agar, glycerin agar, glucose agar, lactose agar. Peptone Water. Derivatives : Glucose peptone water, lactose peptone water, sucrose and mannite peptone water. MacConkey's Media. — Bile-salt litmus glucose peptone water ; bile-salt neutral-red lactose agar. Other Media. — Milk, potato, blood serum, ascitic fluid, urine, whey, gelatin agar, beer wort, bread, eggs ; nitrate media, synthetic media ; animal tissues, etc. Nutrient Broth. — 500 grm. of lean beef finely minced are steeped in one litre of ordinary water for twenty-four hours in a cool place. The fat particles are then skimmed off, the fluid strained off, and the juice well pressed out. This is then boiled for half an hour to coagulate the albumins, filtered, and the bulk made up to 1 litre. One per cent of Witte's peptones and \ per cent of common salt are then added and dissolved by the aid of heat. The broth is now tested as to its reaction, and is usually acid. Its acidity is determined by taking 5 c.c, diluting to 50 c.c. 152 PUBLIC HEALTH BACTERIOLOGY with water, adding i c.c. of phenolphthalein, and titrating with N/io NaOH. Boil one minute before titration. Calculate the amount of N/i NaOH required for the litre of broth made. The convention at present in use is to leave the broth acid to phenolphthalein to the extent that i c.c. of N/i NaOH is required to neutralize ioo c.c. of broth, or 10 c.c. per litre, and the medium is said to be " acid + 10," or + i per cent. Therefore add the calcu- lated amount of N/i NaOH less 10. (Some add the full amount of soda and then make acid to the desired extent with N/i HC1.) Heat to boiling and test again ; if it needs a further correction, boil again. Allow to cool to bring down the precipitate of MgAm phosphate caused by the change of reaction. Filter, place in flasks or tubes (about 5 c.c), and sterilize in autoclave at 1200 C. for 15 minutes, or at 1300 C. for 1 minute, or for 15 to 60 minutes on three successive days in a steam sterilizer at ioo° C. Broth can also be made from Liebig's Extract of Meat, using o*5 per cent of it instead of mince-meat. The other procedure is similar. Neutralization can also be effected by adding saturated solution of NaOH until red litmus is just turned blue. Meat Extract ,or Fleischwasser can be used as a basis for broth and the media derived from it. It is made by warming 500 grm. of minced beef or horseflesh with 1 litre of water at 500 C. for half an hour, and then boiling for half to three-quarters of an hour. Filter, strain, make up to 1 litre, and then pour into a flask ; if not to be used at once, sterilize. Broth contains some muscle sugar or inosite, and to get rid of this is at times inoculated with a young culture of B. coli and incubated at 370 C. for 18 hours, and then boiled to kill the organisms. Glucose and Lactose are added to sugar-free broth (usually 1 per cent). Such media are not sterilized in the autoclave but in a steamer, because the sugars are not stable at high temperatures. Nutrient Gelatin is made from broth by adding 10 per cent in winter and 15 per cent in summer of " gold label" gelatin. Heat on water-bath (as little as possible) to dissolve ; readjust reaction (gelatin makes the medium GENERAL PRINCIPLES 153 acid), filter, and sterilize in the steamer. If filtrate is not clear, cool to 6o° C, add whites of two eggs per litre, re-heat for half-an-hour in steamer, and filter through Chardin paper in warm filter-jacket. Nutrient Agar. — Add 1*5 per cent of powdered agar to broth. Melt in the steamer at ioo° C. for 1-5 hours. Standardize, and replace in steamer for 20 minutes to precipitate phosphates. Cool to 6o° C, add two whites of egg per litre, reheat for half an hour, filter through Chardin paper by aid of a hot-water funnel or in a steamer, or filter through glass-wool. Tube, and sterilize. Agar melts between 900 and ioo° C. and remains fluid down to 400 C. Glycerin Agar. — Add 6 per cent of glycerin after filtration ; tube, and sterilize. Glucose and Lactose Agar. — To agar made with sugar- free broth, add 2 per cent of glucose or lactose. If to be tinted with neutral red, add before filtration 2 per cent of a solution of neutral red (J per cent). Litmus Lactose Agar. — Add to nutrient agar prepared from sugar-free broth 1 to 2 per cent of lactose, and sufficient litmus to give a good colour (about 5 to 10 c.c. of a 1 per cent litmus solution per 100 c.c. of total medium). Conradi and Drigalski's medhim is similar, plus nutrose and crystal- violet. Peptone Water, — Dissolve by the aid of heat 1 per cent of peptones and J per cent of NaCl in distilled water. Tube, and sterilize. For water investigations it is usual to keep a stock solution ten times this strength. Glucose, lactose, sucrose, and mannite are used with peptone water plus Durham's fermentation tubes. Mostly in 1 per cent strength. MacConkey's Media are much used in water examina- tions in this country. Bile-salt Litmus Glucose Peptone Water is made in single strength, and in triple strength thus : — Peptone, 20 or 60 grm. ; glucose, 5 or 15 grm. ; taurocholate of sodium, 5 or 15 grm. ; litmus solution (10 per cent sterile) 100 c.c, and water to 1 litre. Put peptone, glucose, bile-salt, and water in a flask and heat in steamer for 45 minutes. Filter through Chardin's paper, add the filtered litmus 154 PUBLIC HEALTH BACTERIOLOGY solution, place in tubes, and put in Durham's fermentation tubes. Steam for 45 minutes on two successive days. Double strength is also used. In tubing put 10 c.c. of single strength, 10 c.c. of double, and 50 c.c. of triple, into suitable tubes. To these are added respectively 1 c.c, 10 c.c, and 100 c.c. of the water sample. Bile-salt Neutral-red Lactose Agar is composed of agar 20 grm., peptones 20 grm., lactose 10 grm., bile-salt 5 grm., neutral-red aqueous sterile solution (1 per cent) 4 c.c, and water to 1 litre. Dissolve the agar, peptones and bile-salt in 500 c.c. of water by heating in the steamer for 90 minutes. Add rest of water, cool to 6o° C, add the white of one egg, heat in steamer for 45 minutes, filter through a moistened Chardin filter-paper in a warm filter- jacket, heat filtrate in steamer for 15 minutes, add lactose and neutral red, put in tubes, and steam for 30 minutes on two successive days. The medium requires no alkali. Other Media. — Milk. — Fresh milk free from preservatives, with the cream removed, and giving an amphoteric reaction to litmus, is poured into tubes and heated in the steamer for three successive days at ioo° C. (Above no° C. browns it.) To prove sterility, incubate for at least three days at 370 C. before using. Litmus Milk. — Add sufficient litmus to colour. Potato. — Scrub and wash a potato ; bore a cylinder- shaped piece ; split it diagonally and put each half into a sterile tube, with a pad of wool at the bottom, and half an inch of aq. dest. Plug, and sterilize at ioo° C. on three days. Blood Serum. — Collect blood in a sterile cylinder and allow to clot. Set aside for twenty-four hours in an ice chest. Pipette serum into tubes and place these in a sloping position in inspissator at 750 C. for one hour. Repeat on two successive days. When cool, incubate for twenty-four hours at 370 C, and if no growth, they may be considered sterile. Litmus Whey (Petruschky's). — Mix milk with equal quantity of water, heat to 400 to 500 C, and add dilute HC1 to precipitate casein. Filter, neutralize with NaOH, and heat for one or two hours in steamer, filter till GENERAL PRINCIPLES 155 clear, and if necessary neutralize again. Add sterile litmus until a violet hue is produced. Tube, and sterilize. A good medium for observing change of reaction. STERILIZATION AND DISINFECTION. i. Dry heat : (a) Bright red heat of flame : for platinum needles. (b) Dull red heat of flame : for knives, glass rods, etc. (c) Hot air : 1700 C. for 1 hour : for glass-ware and cotton-wool. 2. Moist heat: (a) Boiling in water at ioo° C. : for 5 minutes kills all non-sporing forms: for ij hours kills spores also. (b) Steam at ioo° C. : Koch's steam sterilizer : 1 J hours' full steaming, or 15 minutes' full steaming on three successive days : used for all media. (c) High-pressure steam : in autoclave: atii5°C, 2 minutes for germs, 15 minutes for spores. Never used for gelatin media, or will not re- solidify. Never used for carbohydrate media, as decomposed into other sugars. 3. Chemicals: 5 per cent carbolic, o-i per cent per- chloride of mercury, etc. Allow to remain in con- tact for half-an-hour. Discontinuous sterilization at 57°-75° C. is used for media, like blood serum, that are changed at higher tem- peratures. The object is submitted to 60 minutes' heating, and kept at 200 to 370 C. until next day, when the heating is repeated, and the same procedure repeated for 3 to 8 days. This is to cause spores present to assume the vegetative form and then to kill the same on re-heating. CULTURAL METHODS. i. Inoculation of liquid media, solid media, living media. 2. Isolation of pure cultures by (a) serial inoculation ; (b) plate cultivation (serial dilution) ; (c) differential 156 PUBLIC HEALTH BACTERIOLOGY sterilization ; (d) aerobic and anaerobic cultivation ; (e) deterrent media ; (/) favouring media ; (g) inoculation into susceptible animals. 3. Preparation of toxins, vaccines, and sera. 4. Post-mortem examination of bodies and tissues. 5. Examination of blood, pus, sputum, urine, cerebro- spinal fluid, exudates, and dust, air, water, milk, sewage, soil, shell-fish, water-cress, etc. MODES OF STUDY. Cultures. — Growth on or in various media ; liquefaction of media ; gas production ; acid or alkali production ; indol formation ; colour formation (pigment) ; colour reduction ; proteinchrome formation ; sulphuretted hydrogen produc- tion ; phosphorescence ; nitrate reduction ; toxin formation ; ferment production and effects. Morphology. — Form, motility, flagella, sporing, pleo- morphism, colour, staining reactions, capsulated. Resistance to desiccation, dry heat, moist heat, chemical agents, sunlight, ultra-violet rays. Optimum Temperature for growth, and toxin and ferment formation. Pathogenicity for (a) man, (b) animals, (c) plants. Products of Growth in Host and Culture — toxins soluble and insoluble, ferments. Habitat. Immunity. — Mode of production ; antitoxins, alexins, complement, phagocytosis, opsonins, amboceptors, anti- bodies, agglutinins, precipitins, aggressins. Anaphylaxis (from Gr. " against protection ") — opposite of Immunity. — A state of excessive susceptibility induced in animals by the injection of certain substances (blood serum, white-of-egg, milk, etc.) CULTURAL REACTIONS. Inoculate various media and observe results from day to day on incubation at 200 or 370 C. (as directed). Label tubes with name of organism and date of inoculation, or mark with pencil. GENERAL PRINCIPLES 157 Broth. — Inoculate from agar cultures three broth tubes, one with each of the following bacilli : B. fluorescens liquefaciens, B. subtilis, and B. mycoides. Incubate at 20° C. and examine in twenty-four or forty-eight hours. Culture of B. fluorescens liquefaciens, quite turbid, or " universal turbidity " ; of B. subtilis, quite clear but scum is formed ; of B. mycoides, quite clear but deposit is formed. Gelatin. — Three forms of culture : (i) slant or streak, (2) stab, (3) shake. 1. Slant or streak — for non-liquefying organisms. Inoculate sloped tubes with B. coli communis and Torula alba. Incubate at 200 C. and examine after forty-eight hours. B. coli communis, spread over surface ; Torula alba, growth limited to line of inoculation. 2. Stab culture — to observe presence or absence of liquefaction. Inoculate gelatin tubes by stabbing with B. mycoides, B. megatherium, and Vibrio Finkler-Prior. Incubate at 200 C. and examine after twenty-four, forty- eight, and seventy-two hours. B. mycoides, horizontal liquefaction ; B. megatherium, funnel of liquefaction medium width ; V. Finkler-Prior, funnel of liquefaction wide. 3. Shake culture — to observe gas formation. Melt gelatin at 400 C. on water-bath and inoculate as in the case of broth. Place in rack, and allow to solidify. Incubate for forty-eight hours. Use B. coli communis and B. subtilis. B. coli communis, gelatin full of gas bubbles ; B. subtilis, no gas formed. Agar. — Inoculate sloped agar tubes with the following germs, incubate at 370 C, and examine after twenty- four hours. Results should be as follows : — B. subtilis, dry myco- derma ; B. mycoides, fine filaments ; B. megatherium, confluent moist raised growth ; B. proteus, thin transparent growth over whole surface. Potato. — Inoculate, incubate at 37° C, arid examine in. twenty-four hours. 158 PUBLIC HEALTH BACTERIOLOGY B. subtilis, flesh-coloured mycoderma ; Streptococcus, invisible growth ; B. megatherium, a yellow, raised, moist growth. Blood Serum. — Inoculate, and incubate at 370 C. for two to three days, and examine. B. coli communis, serum solid ; B. pyocyaneus, serum liquefied. Milk. — Inoculate, incubate at 370 C. for forty-eight hours, and examine. B. coli communis, milk clotted and acid ; B. denitri- ficans, no clot, alkaline ; B. pyocyaneus, casein pre- cipitated and partly dissolved ; Streptococcus, no clot, -slightly acid. MacConkey's Broth with Durham's tubes. Inoculate, incubate at 370 C, and examine daily. B. coli, acid and gas ; B. typhosus, acid, no gas. Peptone Water. — Inoculate, incubate at 370 C, and examine in four or five days. B. coli communis, indol formed ; B. typhosus, none ; Sp. cholerae, nitroso-indol. Aerobic and Anaerobic. — Make gelatin stabs and incubate'at 200 C. for two days. B. zopfii, growth only on surface (strict aerobe). Torula alba, on surface and in depth (aerobe and facultative anaerobe). B. butyricus, growth only in depth (strict anaerobe) . Colour Formation. — 1. Inoculate two agar tubes with B. prodigiosus, and incubate ; (1) at 370 C., (2) at 200 C. Examine in forty- eight hours. (1) is white or grey, (2) is pink. 2. Presence of oxygen is necessary in most cases for pigment to be developed. Make two gelatin stab cultures of Bacillus fluorescens liquefaciens. Incubate one aerobically and the other anaerobically. Examine in forty- eight hours. The aerobic culture is pigmented, the other is not. 10 3. A few require absence of oxygen. Make gelatin stab of Spirillum rubrum and incubate at 200 C. for 3 to 4 days, when growth is found in depth to be red and on surface to be white. GENERAL PRINCIPLES 159 Colour Reduction is measured by the addition of some easily discoloured substance to the medium. Litmus, methylene-blue, and sodium sulphindigotate are used. As the bacteria grow, the colour is discharged in the anaerobic parts of the culture. In a fluid medium, shaking restores the colour. Proteinochrome formation is observed in 5 per cent peptone broth or 3 per cent peptone water. Add a few drops of acetic acid and then fresh chlorine water, when a red-violet colour indicates proteinochrome formation. Test for Indol Formation. — To a pure culture in broth or peptone water add 1 c.c. of o-oi per cent sodium nitrite solution and 1 c.c. of purest sulphuric (25 per cent), or HC1. A red coloration within five minutes indicates that indol is present. A second test which gives a positive result with B. coli within forty-eight hours at 370 C. is to add 1 c.c. of an acid solution of paradimethylamido- benzaldehyde, when a rose or cherry-red colour develops in 2 or 3 minutes if indol is present. (Solution A. : para. 8 grm., HC1 160 c.c, absolute alcohol 760 c.c. ; Solution B. : cold saturated solution of potassium sulphate. Use 1 c.c. of A, and shake, and add 1 c.c. of B. and allow to stand.) If there is any suspicion of the micro-organism having the power of reducing nitrates to nitrites, add the sulphuric acid first and wait ; if no coloration develops, then add the nitrite solution. Spirillum cholerae has this power, and hence the addition of the sulphuric acid is alone required. This is called the nitroso-indol reaction. LIQUEFACTION OF GELATIN. This is due to the development of enzymes from the growth of bacteria in proteid media. These proteolytic enzymes are not always secretions of the bacterial cell, but are in some cases closely bound to the cell-body, and are separable from it only after its death. When they are true secretory products, they can be separated from the micro-organisms by filtration through a Berkefeld filter candle, and from such filtrates they can be obtained in 160 PUBLIC HEALTH BACTERIOLOGY the dry state by precipitation with alcohol. Such enzymes are usually more thermostabile than when in solution. Thus, most enzymes are readily destroyed in solution at 700 C, but dry enzymes may withstand 1400 C. for 10 minutes. (As usual, moist heat is more effective than dry heat.) This proteolytic (protein-splitting) or peptonizing power varies for the different proteids, and is usually tried on gelatin, blood fibrin, and casein of milk. Thus, Staphylo- coccus pyogenes liquefies gelatin and blood-serum, and clots milk, but does not dissolve the casein ; Streptococcus pyogenes does not liquefy gelatin, nor blood serum, nor casein ; B. coli communis is likewise negative to all three tests ; some varieties of B. proteus are positive to all three ; B. pyocyaneus is positive to the three ; Spirillum cholerae liquefies gelatin and blood serum, but not casein ; and so on. These tests are still very useful in dividing the bacteria into groups, and so narrowing the field in the difficult task of concluding, with moderate certainty, the race of a particular germ. Gelatin — liquefying Gelatin — non-liquefying Staphylococci B. anithracis Streptococci Pneumococci B. tetani and botulinus M. tetragenus and melitensis B. enteritidis sporogenes B. oedematis maligni Sp. choleras and most Sp. Colon -typhoid group B. diphtheriae B. mallei B. cloacae B. proteus B. subtilis B. pestis Friedlaender's pneumobacillus Yeasts (most) B. pyocyaneus Actinomyces Moulds (most) Note. — Organisms which do not grow on gelatin or at air temperature cannot be thus classified. HEMOLYSIS. Haemolysis will be treated of under immunity, but the present reference is to the haemolytic action of certain organisms when grown on blood-agar plates. (Blood agar is made from defibrinated blood 1 part, and agar 2 parts.) GENERAL PRINCIPLES 161 In such a medium, haemolysis (destruction of the red blood-cells) is shown by a yellow transparent halo around the colonies. Organisms producing hemolysins, are : — Staphylococci, Streptococci, some Spirilla (but not Sp. cholera). STAINING REACTIONS AND METHODS. Saturated alcoholic solutions are kept as stock, and diluted i in 10 with water as required, and filtered. Rather use dilute stains and for a longer time, than have precipitate of stain on preparation. Stains can be reduced in intensity if necessary by using dilute acids, commonly acetic i per cent. Acid stains, like eosin, stain the proto- plasm of cells, whereas basic stains, like gentian-violet, methylene-blue, and fuchsin, stain the cell-nuclei and bacteria. Blood, pus, and smears from agar plates stain most sharply with methylene-blue, but stain fades rapidly on keeping. Certain bacteria need special stains. Loeffler's Methylene-blue. — Saturated alcoholic solution methylene-blue 30 c.c. ; solution KOH (o-oi per cent) 100 c.c. Keeps well. Aniline Oil-Water Stains. — Made with saturated alco- holic solutions of gentian-violet and fuchsin, which are mixed 1 in 10 of aniline oil-water. The latter is a mixture, made by shaking 5 c.c. of aniline oil in 100 c.c. distilled water ; filter, and keep in dark. These keep badly. Carbol- fuchsin (Ziehl-Neelsen) . — Ac. carbolic (5 per cent) 100 c.c. ; saturated alcoholic solution fuchsin 10 c.c. Diluted 3 to 4 times it stains more slowly but better. Keeps well. Carbol-glycerin- fuchsin. — Fuchsin 1 grm., ac. carbolic liq. 5 c.c, glycerin 50 c.c, and aq. dest. 100 c.c Dilute in use 4 to 10 times. Keeps well. Carbol-methylene-blue. — Methylene-blue 1-5 grm., abso- lute alcohol 10 c.c, ac carbolic (5 per cent) 100 c.c. Keeps well. To Make a Film. — Take a cover-slip (in Cornet's for- ceps), and put on it a drop of distilled water (small for fear of plasmolysis) . Take two strokes of culture with platinum needle and rub into drop, and spread out. Dry in air 11 162 PUBLIC HEALTH BACTERIOLOGY (should dry at once). Fix by passing three times through the flame. Stain for two or three minutes. Wash with water and examine on clean slide in water-drop (using oil immersion). In water-drop bacteria look larger, can be restained, and can be kept longer than when mounted direct in Canada balsam. To preserve : allow to dry, remove from slide, roll up, and label. Counterstain with eosin or other stain in dilute solution for i to 2 minutes. Eosin stains cell protoplasm red. Films are also made on slides, which are more easily handled than coverslips. Gram's Method of Staining — Depends on the fact that some bacteria when well stained retain the stain after treatment with a solution of iodine and subsequent washing with alcohol (strong or absolute). This is believed to be due to the stain and the iodine forming a combination which resists decoloration. Table. Gram-positive Gram-negative (Retain the gentian-violet) Staphylococcus pyogenes Streptococcus ,, Pneumococcus (Take the counterstain) Meningococcus Gonococcus Micrococcus melitensis Micrococcus tetragenus Bacillus anthracis subtilis diphtheriae tetani catarrhalis Colon-typhoid group of bacilli Cholera group of spirilla Bacillus pestis and group ,, mallei botulinus influenzas tuberculosis and other acid-fasts aerogenes capsu- latus pyocyaneus ,, proteus „ Koch- Weeks Morax - Axenf eld „ enteritidis sporo- genes of swine erysipelas maligni cedematis anthracis symptomatici of fowl cholera of mouse septi- ,, caemia ,, of potato Yeasts and many moulds of rabbit septicaemia Spirillum Obermeieri (spirochaete) Friedlaender's diplobacillus Streptothrix actinomyces Method. — Stain for 5 minutes with aniline-oil-gentian- violet, or carbol-gentian-violet. Pour off excess of stain GENERAL PRINCIPLES 163 and cover with Lugol's (or Gram's) solution of iodine (iodine I, KI 2, aq. dest. 300) for 30 seconds to 2 minutes. Wash with 97 per cent alcohol (or methylated spirit) until washings are no longer coloured (takes about 30 sec. to 2 min.). Examine in water, or dry and mount in balsam. To counterstain : remove alcohol with water and cover with dilute carbol-fuchsin for a few seconds (or saturated watery solution of Bismarck brown for longer). Wash in water, dry, and mount. Result : Bacteria blue-black, or colourless, or red ; tissues red. Those bacteria which are blue-black are said to be Gram-staining or Gram- positive. The others are said to be Gram-negative. (See Table on previous page.) Acid-fast Bacteria. — Some bacilli stain with difficulty with ordinary dyes, requiring the aid of heat or a mordant (as carbolic). Such bacilli usually retain the stain even when treated with dilute acids and alcohol, and hence are called " acid-proof " or " acid-fast." This resistance is believed to be due to the presence in the cell-body of a waxy substance (an alcohol). The members of this group are : Bacilli of human, bovine, avian, and fish tubercu- losis; Moeller's Timothy-grass bacilli (1) and (2) ; Mist- bacillus; Rabinowitch's butter bacillus; Korn's butter bacilli (2) and others ; Johne's bacillus (of chronic bovine pseudo-tuberculous enteritis) ; Bacillus smegmatis (smegma bacillus); Bacillus leprae (leprosy bacillus). Method. — Flood slide or cover-glass with carbol-fuchsin and heat for 3 minutes. Wash and decolorize by dipping into 5 per cent sulphuric acid and 60 per cent alcohol alter- nately until film looks colourless. Wash in water. Counter- stain with aqueous methylene-blue for a half to one minute. Wash and examine. The acid-fast bacteria are stained red, while the others and the matrix are stained blue. Alcohol-fast Bacteria. — In specimens of urine being examined for tubercle bacilli, acid-fast smegma bacilli may also be present. To distinguish: counterstain film in a saturated solution of methylene-blue in absolute alcohol for 5 minutes. Tubercle bacilli remain red, while smegma bacilli become blue. Capsule Staining. — Many bacteria possess a mucoid or gelatinous envelope, though it is only in a few species 164 PUBLIC HEALTH BACTERIOLOGY that it is easily demonstrable. It is known as the " capsule " and varies in thickness from being only just visible to 4 or 5 times the size of the bacterium itself. It is mostly seen in preparations taken directly from animal tissues or fluids or exudates, or from cultures in media containing animal serum or milk. It is best seen in the Diplococcus pneumoniae, Micrococcus tetragenus, B. aerogenes capsulatus, and the bacilli of the Friedlaender group. Hiss's method : Make a cover-slip film, and preferably by using a drop of animal serum instead of water. Dry in air and fix by heat. Stain for a few seconds with dilute fuchsin or gentian-violet (1 of saturated alcoholic solution in 19 of aq. dest.), meanwhile heating the preparation over a flame until steam arises. Wash off dye with 20 per cent watery solution of copper sulphate. Blot dry (do not wash with water), and mount direct in Canada balsam. The capsule appears as a faint blue halo around a dark purple cell-body. Spore Staining. — Prepare film as usual and fix in the flame. Place in CHC13 for two minutes. Wash in water. Place in 5 per cent chromic acid for half a minute to two minutes. Wash in water. Float cover-slip, film side down, on carbol-fuchsin solution in a small porcelain basin and heat stain gently until it steams ; continue in stain for 3 to 5 minutes. Decolorize in 5 per cent sulphuric acid for 5 to 10 seconds. Wash in water. Stain with saturated watery methylene-blue for 30 to 60 seconds. Wash and examine ; or dry, and mount in balsam. The spores are stained red and the cell bodies blue. Spores are believed to be an encysted or resting stage of bacteria, and not a method of reproduction, or rather multiplication. In most cases only one spore is produced by one bacillus, and the latter becomes extinct when the spore is fully developed. Spore formation is not very common among bacteria, and is found almost exclusively among the bacilli, less commonly in the spirilla, and rarely, if at all, in micrococci. The anaerobic bacilli are almost all spore-forming, but amongst aerobes the only sporing bacterium pathogenic to man is the anthrax bacillus. This materially facilitates and simplifies the disinfection and treatment of infectious diseases, as spores are extremely GENERAL PRINCIPLES 165 resistant to injury by heat, light, drying, and chemicals. True spores or endospores are to be distinguished from arthrospores, the existence of which is now seriously questioned. An arthrospore is a bacterium which enters into a resting stage without any new formation within the protoplasm. It stains well with ordinary stains, and has no distinct capsule, but is stated to have increased resistance to external agents. A true spore (i) resists the ordinary staining method ; and (2) shows very great resistance to destruction to the usual agents. Flagella Staining. — Flagella are hair-like organs used for locomotion, and have been described as occurring on bacilli, spirilla, and a few species of cocci. They are best seen in young cultures, 10 to 18 hours old, at 370 C. McCrorie's method gives admirable results when the technique is carefully followed. McCrorie's Flagella Stain. — Measure out and mix : — Night-blue, 1 grm. in 20 c.c. of absolute alcohol ; potash alum, 1 grm. in 20 c.c. of distilled water ; tannin, 1 grm. in 20 c.c. of distilled water. Allow mixture to stand for twenty-four hours, and filter supernatant fluid. Keep stain in incubator and filter again when using. Method. — (1) Take some distilled water at 370 C. in a watch- glass ; place therein a loopful of young agar cul- ture, and allow to swim off and diffuse without stirring. (2) Take several loopfuls of this solution, and deposit them singly, without smearing, on a clean cover- slip. (3) Dry in the incubator at 370 C. (4) Apply stain (also at 370 C.) and replace in incubator for 10 minutes. (5) Wash off stain by dipping cover-slip edgeways several times into water at 370 C. (6) Dry in the incubator. (7) Mount : or counterstain bodies with strong fuchsin solution for 2 minutes ; wash, and dry as before ; mount. Flagella are blue, bacillary bodies are red. 166 PUBLIC HEALTH BACTERIOLOGY Granules. — Diphtheria bacilli when stained show oval bodies, which stain more deeply than the rest of the cell. Loeffler's methylene-blue (page 161) shows them well, but a contrast stain is often used, such as that of Neisser. Neisser's Method. — Two solutions are used : Solution i : methylene-blue I grm. + 20 c.c. alcohol (96 per cent) + 50 c.c. glacial acetic acid + 950 c.c. aq. Solution 2 : Bismarck-brown 2 grm. dissolved in 1 litre of boiling distilled water. Make a film, fix, stain with Solution 1 for 30 to 60 seconds. Wash, and pour on Solution 2, and after 30 seconds wash off with water. Dry, and mount. Bodies of the bacilli are brown, and the granules are blue. Paraffin- section Staining. — Sections must first be fixed on slides by one of two modes : — 1. Float section on warm water (under 400 C.J, insert slide underneath, with a needle fix one corner, and withdraw slide. Dry for 24 hours in incubator at 370 C. 2. Place a drop of solution of egg-white (10 per cent in aq.) on a slide, draw on section as before, and incubate at 370 C. for 30 minutes ; or, remove excess of moisture, heat over small flame until paraffin melts, and then until vapour arises. Staining. General Method. — 1. Preparation : Remove paraffin with xylol, and xylol with absolute alcohol. Wash in water (unless alcoholic solution of stain is used). 2. Staining : Use methylene-blue for 15 minutes ; carbol-thionin-blue (5 minutes), or aniline-oil- gentian violet, carbol-fuchsin, etc. For over- staining reduce with very weak acid for 5 to 30 seconds. This also decolorizes the tissues. 3. Counter-staining : WTash in water, stain with \ per cent eosin for 30 seconds, and wash in water. 4. Dehydration and Clearing : Remove water with absolute alcohol (some organisms are decolorized very easily at this stage, and hence treatment must be rapid). Remove alcohol with xylol, and mount in Canada balsam. Weigert advises aniline oil, aniline-xylol, xylol, and balsam. GENERAL PRINCIPLES 167 Gram's Method. — (i) Prepare ; (2) Stain for 5 minutes with aniline-oil-gentian-violet ; (3) Pour off excess, do not wash, flood with Gram's iodine solution repeatedly, until purplish black, and allow to act for 1 minute ; (4) Do not wash, but decolorize with absolute alcohol or methylated spirit until faint violet tint ; (5) Wash in water, counter- stain with J per cent eosin for 1 minute ; (6) Dehydrate, clear, and mount. Bacteria are blue-black, and tissues are pink. In Weigert's modification of the Gram method, the section is first stained for 30 minutes in lithia-carmine. Wash in water and proceed as in Gram's method, except that the dehydration is done with aniline oil. Acid- fast Bacilli in sections. — Tubercle bacilli, etc. (1) Prepare ; (2) Stain in carbol-fuchsin for 5 minutes in hot. solution, or 24 hours in cold ; (3) Wash in water ; (4) Decolorize in 12 per cent sulphuric acid ; (5) Wash well in water (colour should just be a faint pink) ; (6) Contrast stain with saturated watery solution of methylene- blue for 30 seconds ; (7) Wash in water, dehydrate, clear, and mount. Bacilli are red, tissues are blue. Note. — If a section has been hardened in corrosive sublimate, the latter must be removed after the paraffin. This is done by using equal parts of Gram's solution and water for a few minutes, and then removing the iodine with methylated spirit. POLYCHROME STAINS. These are of value for the staining of micro-organisms in pus and exudates, and for blood films, in all of which the relation of the bacteria or protozoa to the cellular elements is to be determined. In all these stains the basis is a mixture of solutions of methylene-blue and eosin, which stain the various elements separately and in combination, thus bringing out in a marvellous way the details of the structural and foreign bodies. This mixture is called the Romanowsky stain, and various modifications of it are now in use. Jenner's Stain. — A simple stain, excellent for blood work, but not so good for parasites as others given below. No 168 PUBLIC HEALTH BACTERIOLOGY alkali is used in its preparation. Equal parts of watery solutions of (a) Gruebler's water-soluble eosin (i-2 per cent), and (b) Gruebler's medicinal methylene-blue (i per cent), are mixed, and the mixture allowed to stand for twenty- four hours. A coarse granular precipitate forms, is filtered off, dried at 550 C., washed with distilled water, filtered, washed again, filtered, and dried. Of the dried powder, 0-5 grm. is dissolved in 100 c.c. of Merck's methyl alcohol. In use, a few drops are placed on the dried unfixed film for one to three minutes, then poured off, and the slide is washed with distilled water until pink in colour. Dry with filter-paper, and mount in xylol balsam. Leishman's Stain. — The methylene-blue is alkalinized with 0-5 per cent of sodium carbonate, weaker eosin oolution is used, and the technique of preparation is varied. The stain will keep for a long period. In use, a few drops are placed on the unfixed preparation for fifteen to thirty seconds, the film being tilted from side to side to prevent drying at any part. In this way the film is both fixed (by the methyl alcohol) and stained by one operation. About twice as much distilled water is now added, and the diluted stain allowed to act for five minutes longer. Now wash in distilled water, mount, and examine. Giemsa's Stain and Method. — This is a modified Romanowsky, of great value in staining Spirochseta pallida, Vincent's spirilla, protozoa, and Negri bodies. The stain used is methyl-azure, which Giemsa believes to be the essen- tial constituent of the Romanowsky stain. In use, the film is first fixed with alcohol, dried, covered with the stain, dilu- ted, well washed, drained, dried, and mounted. For ordinary staining, fifteen minutes are enough ; for the spirochete and Negri bodies, one to twelve hours may be necessary. Staining. — By Romanowsky, red cells are stained orange to pink ; eosinophile granules, red ; neutrophile granules, yellow to lilac ; nuclei, shades of violet ; blood platelets, purplish ; malarial parasites, blue ; chromatin, red to rose-pink. Blood Films. — May be made on cover-slips or on slides. With cover-slips, touch one to the exuding blood, drop it on another, and then draw the cover-slips apart. For slides, touch a drop of blood near one end of slide, and smear GENERAL PRINCIPLES 169 out with another by drawing it slowly along the first, sloped to it at an angle of about 450. The slides used must be clean, and are usually stored in absolute alcohol, which is burnt off just when using. When the film is stained, it can be examined at once, with a drop of cedar oil, and afterwards mounted, if desired. Fixation is accomplished by methyl alcohol after air- drying, when using the Romanowsky method. For other processes it is attained by one of the following methods : — (a). Half an hour in a hot-air chamber at 1200 C. (b). Half an hour in a mixture of alcohol and ether (equal parts). Wash, and dry. (c). Five minutes in formol-alcohol (1—9). Wash, and dry (Gulland). (d). Two to three minutes in saturated solution of corrosive sublimate. Wash well, and dry. For wet films, which give the histology better, the methods are varied. The films, while still wet, are placed film downwards in the fixative. Gulland's combination of (b) and (d) is said to be an excellent one. INOCULATION OF ANIMALS. Inoculation of animals, or the animal experiment, as it is called, is used for a variety of purposes, is made in a variety of ways, and the number of different animals used is now considerable. It is an important way of getting a pure culture in difficult cases. It also determines the pathogenicity of a pure culture injected. By the occurrence of special symptoms following injection of suspected material, it serves to establish the presence of a particular micro-organism in the material. If the injection of known products of certain organisms is followed by certain reactions, the presence in the animal's body of the organism from which the product has been derived is inferred (tuberculin and mallein tests) . By passing a par- ticular organism through a series of susceptible animals in succession, its virulence may be exalted, and the same experiment through resistant animals may depress its vitality ; this is Pasteur's " method of passage." Animal inoculation is also used for the production of anti- toxins and antibacterial bodies. 170 PUBLIC HEALTH BACTERIOLOGY The inoculation may be : (i) Cutaneous, that is, rubbing into the unbroken skin ; (2) Subcutaneous, with a syringe, or by cutting the skin and putting the material in a pocket in the subcutaneous tissue and stitching up the skin wound ; (3) Intraperitoneal ; (4) Intramuscular ; (5) Intrapleural ; (6) Intravenous ; (7) Into the stomach by a tube, or by ordinary feeding ; and (8) By inhalation. The various processes are described as required under the particular microbes concerned. The average tempera- tures of the more commonly used animals and a few others may be here conveniently tabulated. The table is compiled from Abel's " Laboratory Handbook of Bacteriology," and from various other sources. Table of Animal Temperatures. Rectal Temperature Pulse Centigrade Fahrenheit Rate Where usually observed Guinea-pig 37-3° to 39-5° 99° to 1030 — Horse 37'7° to 38-3° ioo° to IOI° 35 to 45 jaw Cow 377° to 38 90 IOO° to 102° 45 to 55 jaw Calf 38-4° to 39-9° 1010 to 103-8° — — Sheep 38-8° to 40° 102° tO IO40 70 to 80 heart Pig ditto ditto ditto ditto Goat 38-6° to 3970 ioi#4°to 103-4° — — Dog 38-6° to 39'5° 101-4° to 103° 80 to 90 — Cat 377° to 38-3° ioo° to IOI° — — Rabbit 38-3° to 39-9° ioi° to 104° — — Chicken 410 to 42-5° 105° to 108-5° — — Pigeon ditto ditto - — Linnet 44° 111° — — Rhesus Monkey. . 38-1° to 39-5° 100-5° to 103° — — Chimpanzee 37° to 380 98-4° to 100-4° — — Bat 4i 106° — — Narwhal . . 35-5° 96° — — Reptiles . . 28° 82-5° — — UNICELLULAR MICRO-ORGANISMS. Fungi. — Fungi are members of the class of plants called Thallophyta, which show no division into root and stem. They are distinguished from the algae of the same class by not possessing chlorophyll. GENERAL PRINCIPLES 171 Schizoniycetes, or fission-fungi — multiply by fission : coccus, bacillus, spirillum, streptothrix. Blastomycetes, or budding-fungi — multiply by budding: the yeasts or torulae. Hyphomycetes , or branching-fungi — multiply by branch- ing. The branches are called hyphae, and the network of interlacing threads is called the mycelium : the moulds. Protozoa. — Unicellular members of the animal kingdom. They are divided into groups like the following : — Sarcodina. — Naked or cased organisms which throw out pseudopodia : amceba. Flagellata. — Endowed with organs of locomotion — flagella : trypanosoma, spirochaeta. Infusoria. — Locomotion by means of short flagella, called cilia, of which many are present : balantidium. Sporozoa. — Non-motile in adult state. Reproduce by spores. Feed by osmosis. Exclusively endoparasites : Plasmodium malariae or haemosporidia, piroplasma, coccidium. Schizomycetes may be classed in various ways under the following heads : — Parasites or Saprophytes Aerobes or Anaerobes The parasites, saprophytes, aerobes, and anaerobes may be either obligatory or facultative. Pathogenic or Non-pathogenic Sporing or Non-sporing Motile or Non-motile Flagellated or Non-flagellated Gelatin-liquefying or Non-liquefying Gram-staining or Non-Gram-staining Chromogenic or Non-chromogenic. Other characters used to distinguish bacteria into groups are : their action on the various sugars, the production of indol in certain media, reduction of nitrates, their behaviour to the dyes, e.g., acid-fast or not, polar staining, etc. CHAPTER X. RESULTS OF BACTERIAL ACTIVITY. PRODUCTS. These result mainly from the cleavage of proteids and fats, and the fermentation of carbohydrates. The basis of our knowledge on this subject was laid by Pasteur, who also was the first to prove the part played by micro- organisms in these processes. The actual work. of cleavage is carried out by ferments or enzymes. A ferment or enzyme is a substance produced by a living cell, which substance is able to bring about enormous chemical change (in proportion to its bulk) without itself suffering decomposition. The accumulation of its products often causes its action to cease, but if these are removed, the action is indefinitely prolonged. We shall see that the toxins of bacteria have been compared to enzymes, and while to some extent there is a resemblance in their action, the toxins in a certain amount are able to produce only a definite result, which is less than that produced by a larger dose. The various enzymes are grouped as proteolytic (in culture, gelatin-, fibrin-, serum-liquefying), fat-splitting, and carbohydrate - splitting (produce alcohol, simpler sugars, lactic acid, butyric acid, acetic acid). Other activities are : denitrification, nitrification, light- production, colour-production, sulphur-utilization (sulphur bacteria), etc. Ptomaines. — The action of bacteria on dead animal mat- ter by their proteolytic enzymes, produces substances called ptomaines, or " animal alkaloids." These bodies are toxic to the human species (and others) , and are organic chemical compounds, basic in nature, which combine with acids to form salts. They have to be distinguished from leuco- maines, similar substances formed in the living body during proteid metabolism, and not by bacterial action. They have also to be distinguished from the bacterial toxins, BACTERIAL ACTIVITY 173 which are developed by bacterial growth, independent of the medium in which grown, and have even been obtained in cultures in proteid-free media. Of the ptomaines, putrescin and cadaverin are extremely poisonous, and most cases of meat-poisoning, cheese-poisoning, and vegetable- poisoning are due to one or another of these ptomaines. INFECTION. The invasion of the animal body by bacteria is spoken of as infection if it gives rise to disease. The definition requires extension to cover the case of diphtheria, where the invasion by the micro-organism is often very slight, but where the disease is due to the invasion of the body by the toxins or bacterial products. In most cases the infection is due to both the bacteria and their products, in varying degrees. In the first place it is useful to note that the skin and the mucous membranes of the alimentary tract, the mouth, the nasal passages, the upper respiratory tract, the con- junctivae, and the genital passages, are normally inhabited by various species of bacteria. Some of these are facultative parasites, and seize the opportunity of a break in the surface, or other injury, to grow and multiply, and so produce disease. Others are pure saprophytes, non- pathogenic in any circumstances to the body on which they harbour. The definition of the infective diseases will be useful here. An infective disease, or rather a specific infective disease, is one which results from the introduction into the body, (i) by wounds, (2) by the air-passages, or (3) by the alimentary tract, of a definite ferment, or poison, or micro-organism, which grows and multiplies in the body. In some of these diseases the poison is given off again, and they are then spoken of as infectious, or transmissible from person to person. Where contact is necessary for transmission, they are called contagious. The tendency is to give up the use of these terms, infectious and con- tagious, and simply to speak of infective diseases, which are transmissible in various ways, as by the air, by food, by contact, by fomites, by insects. 174 PUBLIC HEALTH BACTERIOLOGY They are called specific, because they have a perfectly definite course, characterized by the stages of incubation, invasion, advance and death, or decline and convalescence. Some of them have also a skin eruption or rash. The infection is given off again by the breath, exhalations from the skin and wounds, by desquamated portions of the epidermis, by the secretions and excretions (mucus of mouth and throat, saliva, sputa, faeces, urine, seminal fluid, milk). Micro-organisms, then, are only relatively pathogenic or non-pathogenic, and in any particular instance of pathogenicity, the amount and kind of attack vary with a large number of factors. This is not a matter for wonder now, with our knowledge of the varied needs and differences in vitality of many micro-organisms, but was a stumbling- block in the early days. Following Muir and Ritchie, we may summarize the matter thus : — Infection is conditioned by (i) the infecting agent, and (2) the subject. 1. The infecting agent produces its effect dependent on (a) its virulence, (b) its numbers, (c) its path of entrance. 2. The subject varies in its susceptibility or the reverse (resistance), according to (a) its species, (b) race, (c) age, (d) individual peculiarities, (e) vitality, (/) other disease. Mode of Action. — Multiplication ; invasion of lymphatics ; invasion of blood-stream ; settlement in certain tissues ; chemical products (toxins), locally or diffused. Effects of Bacterial Action. 1. Tissue changes : — (a) Local — tissue reactions or degeneration and necrosis, acute or chronic. (b) Distant — damage to special tissues, reaction of blood-forming organs. (c). General — malnutrition or increased waste, or both. 2. Metabolic changes : fever, etc. BACTERIAL ACTIVITY 175 BACTERIAL POISONS. The knowledge of these is by no means complete, so that sharp distinctions between various kinds cannot be, at present, depended on. The first to study their pro- duction was Brieger, and it was while so engaged that he was led to the discovery of the ptomaine poisons. These bodies, however, did not, on injection, reproduce the symptoms of diseases associated with the bacteria con- cerned in their production, and so the ptomaines are not nowadays classed as true bacterial poisons. Roux and Yersin, in 1889, filtered broth- cultures of B. diphtherias through unglazed porcelain (Chamberland filter), and showed that the filtrate was bacteria free, and yet on injection the filtrate produced practically the same effects as the injection of the living bacilli. From this it was inferred that the filtrate contained the toxin of the diphtheria bacillus. The same method applied to other bacteria yielded no such result in most, and so the con- ception was reached that some bacteria secrete or excrete poisons which are soluble in the media in which they are grown, and some do not. Of the former class, diphtheria and tetanus are the types ; of the latter, the tubercle bacillus may be taken as a type, but the class is a very large one, including all the bacteria except diphtheria, tetanus, botulinus, and the anaerobes generally (some to only a small extent). Other bacteria, such as dysentery and cholera, are said to produce soluble poisons, but the results are still discordant. As a consequence of these findings, and of the further observation that, in the bacteria not secreting soluble poisons, the injection of dead bacteria could reproduce many of the characteristic lesions of the disease associated with them in the living state, the division of bacterial poisons into two groups has arisen, viz : — 1. Extracellular toxins, true toxins, or soluble toxins. 2. Intracellular toxins, endotoxins. After the removal of these bodies from the bacteria, a certain proteid residue remains, which, on injection, gives rise to localized reactions. Buchner calls this residue " bacterial protein," and believes it to be the same in all 176 PUBLIC HEALTH BACTERIOLOGY bacteria, to be without specific toxic action, but having a positive chemiotactic action on the white cells of the blood, and so preparing the way for the formation of pus. It is still doubtful how much reliance can be placed on the total separation of the soluble and endotoxins from the bacterial protein, and until this doubt is resolved, these conclusions must be accepted with reserve. Extracellular Toxins. — Of the extracellular or true or soluble toxins, that of B. diphtheriae may be taken as the type. As a class they may be defined as the secretory products of the bacterial cells, passing out into the medium, and soluble therein. That the soluble toxins are only so produced has yet to be proved, and in the meantime they may be ascribed to the following sources, which may occur singly, or in any combination : — i. vSecretion or excretion from the bacterium. 2. Action of the bacterium on the medium. 3. Death of the bacterium and liberation of toxins from its disintegrated body. The soluble toxins are easily obtainable in large quantities, nevertheless they have not yet been isolated in a pure form, and so our knowledge of them is derived from the study of the complex filtrates in which they are found. Their action is characterized by being selective or specific for certain tissues ; for example, diphtheria, tetanus, and botulismus toxins all attack the nervous system. In the case of many of them, a definite time elapses before symptoms appear after injection. This has been called a period of incubation, though it is suscep- tible of other explanations. The extracellular toxins are apparently uncrystallizable, are soluble in water, are dia- lysable, are precipitated with proteids by absolute alcohol and by ammonium sulphate, are allied to albumoses, and are relatively unstable to heat, light, and chemicals. Intracellular Toxins. — The endotoxins are either not excreted from the bodies of the bacilli, or are closely bound thereto, or are insoluble in the medium, and remain on filtration on the same side as the bacteria. Any of these theories would account for the known facts in regard to the endotoxins. The greater number of the pathogenic bacteria seem to act by poisons of this class. The poisons BACTERIAL ACTIVITY 177 are Jiberated only after the death of the bacteria, by the breaking up of their bodies, and even then they cannot be obtained apart from the bacterial protoplasm. Their action has therefore been mostly studied by injection of the dead bodies of bacteria. The effects produced are not specific, but are more those of general disturbances of metabolism ; nor does much time elapse before the appearance of the symptoms, that is, there is no so-called incubation time. The intracellular toxins are less sensitive to heat than the soluble ones, but are mostly destroyed by heating to 700 C. The notable exceptions to this are those of the tubercle bacillus, which are still toxic after digestion at ioo° C, and those of the B. enteritidis (Gaertner) which remain toxic after the infected flesh has been cooked. Some organisms, such as B. anthracis, possess no soluble toxin, nor does the injection of the dead bodies induce toxic effects. Yet in the disease produced by the living bacterium, symptoms which suggest toxin action are present. To meet such cases, the hypothesis has been made that these organisms only produce toxins in the animal tissues, or may produce complementary substances which assist the action of endotoxins. Such substances have been studied under the name of " aggressins." An animal is given a lethal dose of an organism, injected into a serous cavity, into which a serous exudation results. On the death of the animal, some of the exudate is taken, most of the bacteria are removed by centrifugalizing, and the few that remain by shaking up with toluol, and allowing to stand for some days. This fluid, on injection, is non- pathogenic, but has the power of increasing the effect of the particular bacterium which has caused its production, so that a non-lethal dose of the bacterium becomes a lethal one ; and not only so, but the fatal effect is more quickly produced. These results are ascribed to a paralysing action of the " aggressins " on the phagocytic functions of the leucocytes. Leucocidin, a true soluble toxin produced by some strains of staphylococcus, causes the death and partial solution of the leucocytes, and this would suggest that the aggressins might after all be toxins, either of the extra- or intra-cellular variety. 12 178 PUBLIC HEALTH BACTERIOLOGY Some bacteria give rise to both varieties, and it is now claimed that this is the case with cholera and dysentery organisms. Nature of Toxins. — The nature of toxins is likewise ill understood. Sidney Martin found that the action of anthrax, diphtheria, tetanus, and ulcerative endocarditis organisms on albuminous bodies was to produce albumoses and peptones, thus resembling the gastric and pancreatic ferments. C. J. Martin, working at the same subject, found that the toxins could pass through a Chamberland filter, the pores of which had been filled with gelatin. From the fact that albumoses can also pass through, it is inferred that the toxins have a molecule of about the same size as the albumoses. Are the toxins of the nature of ferments ? Sidney Martin suggests that the primary toxic agents are of this nature, and by digesting the tissues produce albumoses, which cause the symptoms. The labile nature of the toxins is also urged as a point of resemblance between them and the ferments, as also the so-called period of incubation which follows their injection. If it is a fact that the action of a toxin is strictly propor- tional to its dose, comparison between toxins and the ferments is rendered unnecessary, as this is a fundamental difference ; the so-called resemblances are then mainly fortuitous. Allied Animal and Vegetable Poisons. — Ricin, abrin, robin, and venins. Major Lamb calculates that 0-015 grm- (roughly, J grain) of cobra venom is a fatal dose for a man, which is large in comparison with the minimum lethal dose of tetanus toxin for man of 0-00023 grm. (about ^-^ grain). All these poisons resemble the soluble bacterial toxins, but are less easily dialysable, and hence have been called toxalbumins. The snake poisons are very complex bodies, containing one or more of several toxins, such as neurotoxins, cell toxins, hemolytic toxins, etc. Flexner and Noguchi discovered that the hemolytic toxin of the cobra venom has no action by itself on the red cells, but requires the presence of normal serum. The latter is then said to contain a " complement " which "activates" the venom. Kyes and Sachs farther showed BACTERIAL ACTIVITY 179 that lecithin (a highly complex fat found in the nervous system, and to a less extent in bile) has the property of activating the haemolytic substance in cobra venom. This is very important, since it points to a definite chemical combination, leading to the formation of a toxin from two non-toxic bodies, and is in line with the observed for- mation in diphtheria of an antitoxin, which has many of the characters of a chemical antidote. CHAPTER XL IMMUNITY AND ANAPHYLAXIS. Immunity, or resistance, may be denned as that power or function of the living organism, natural or acquired, which enables it to repel or prevent infection of itself by micro-organisms or their products. Anaphylaxis, or excessive susceptibility (hypersuscep- tibility, supersensitiveness), is defined as a state of extreme sensitiveness to the injection of certain substances, such as bacterial proteins, animal and vegetable albumins (blood serum, egg white, milk), brought about by one injection of the same substances or present from hereditary transmission. Both these terms are relative, in most instances. Thus, birds, while immune from tetanus toxin in any doses likely to result from natural infection, may be killed by enormous doses given experimentally. Similarly, in man, the immunity conferred by one attack of a disease like small-pox, may be overcome in special circumstances of dosage and environment. Absolute immunity does exist. Thus, so far, no animal has been infected with leprosy ; also, cold-blooded animals, under their normal conditions, are absolutely immune to the pathogenic bacteria of the warm-blooded animals. The wild carnivora have a very high degree of resistance to bacteria. Absolute anaphylaxis of a kind also occurs. Thus, the injection (subcutaneously) of 0-25 c.c. of the serum of an eel into a rabbit causes the death of the rabbit in a few minutes. Also the offspring of animals which have been themselves sensitized by injection, show a high degree of anaphylaxis from birth. (The strict use of the term " absolute " would require that the rabbit should die no matter how small the dose of serum used. Thus its use here is relative.) IMMUNITY AND ANAPHYLAXIS 181 IMMUNITY. Immunity may be natural or acquired, and may be considered under the following heads: — Natural Immunity. — Depends on (a) individual, (b) race, and (c) species. Acquired Immunity. — (a) By an attack of the specific disease ; (b) By active immunization with living bacteria, dead bacteria, or with toxins or filtrates ; (c) By passive immunization with antitoxic serum, or antibacterial serum. Natural Immunity, or the resistance to bacteria con- ferred by nature, is a characteristic of the living organism, which varies with the individual, race, and species. Thus, individuals vary greatly in their resistance to infection from slight wounds, from polluted water and milk, and from micro-organisms generally. The young animal is usually less resistant than the mature of the same kind. As regards race, negroes are noted for their high degree of resistance to yellow fever, and in a less degree to malaria, yet they quickly sicken and succumb to small-pox and measles. Among animals, the Algerian sheep are more highly resistant to anthrax than the European races. The influence of species is seen in the non-liability of the human to certain animal diseases, such as cattle plague, fowl cholera, and swine erysipelas, whilst animals are equally resistant to such human infections as cholera, influenza, measles, etc. The causes of natural immunity are usually given as : — (i) The action of certain leucocytes and other cells in engulfing and destroying the bacterial invaders, called phagocytosis, and (2) The action of the blood serum. I. Phagocytosis. — Metchnikoff in 1884 advanced the theory of phagocytosis, based on a careful study of the subject, and since confirmed by many observers. The phagocytes are in part wandering cells, and in part fixed tissue cells. The chief wandering cells are the poly- morphonuclear and large mononuclear leucocytes and wandering tissue cells. Of the fixed phagocytes, possessing the power of amoeboid movement, the cells lining the serous and lymph spaces, the cells of the spleen pulp, and bone 182 PUBLIC HEALTH BACTERIOLOGY marrow, are the chief examples. Metchnikoff calls the poly- morphonuclear leucocytes, Microphages, and all the other phagocytes, Macrophages. He observed the phagocytosis in a fungus disease of a water-flea (Daphnia), and in frogs infected with anthrax, where the death and dissolution of the bacilli could be seen going on inside the phagocytes. Later researches proved that the same phenomena could be observed in all infective processes, more especially if the animal were resistant to the infection. When the micro-organisms get into a part where few phagocytes are, a migration towards the affected spot occurs. This is part of the inflammatory reaction which follows infection. The cause of this migration is the presence of substances in the part, which attract the phagocytes, and is known as positive chemiotaxis. Negative chemiotaxis, or the repulsion of the phagocytes, also occurs. Buchner showed that dead bacteria, bacterial proteins, and closely allied substances, such as vegetable casein (legumin), have a positive Chemiotaxis, whilst the toxins of many virulent bacteria have a negative chemiotactic power. In natural immunity, phagocytosis is developed to a high degree, and it is of such constant and regular occurrence that we may often foretell from the degree of phagocytosis whether, in a particular infection, the animal being experimented with will gain the victory or not. A clinical application of these results is seen in the observation of the increase of the number of leucocytes in the blood during the progress of a disease like pneumonia. The increase is called " Leucocytosis," and is almost wholly of the polymorphonuclear variety. In pneumonia an early and marked leucocytosis is a sign of favourable import, and may be from 12,000 to 40,000 per c.mm. The absence of leucocytosis, except in very slight infections, is highly unfavourable. (It is interesting to note here that whoop- ing-cough gives a Lymphocytosis, as also do enlarged tonsils, rickets, scurvy, and a few other diseases.) 2. The Action of the Blood Serum. — Besides the direct action of the phagocytes, as described by Metchnikoff (the cellular theory), the blood seium was found to have bactericidal power. Von Fodor showed that freshly drawn rabbit's blood could destroy anthrax bacilli, as IMMUNITY AND ANAPHYLAXIS 183 also could defibrinated blood, the pericardial fluid and the aqueous humour, and that this power was lost by heating to 550 C. Buchner (with others) found that completely cell-free blood was bactericidal, and lost this power on heating to 550 C, but not on freezing and thawing. According to Buchner, fresh blood frozen and thawed loses its power because the red cells are destroyed by the process, and make the blood so suitable for bacteria that the bactericidal power is compensated for. These protective substances in the serum are called " Cytases " by Metchnikoff, and "Alexines " by Buchner. These are now believed to be derived from the leucocytes, Metchnikoff holding that they are only formed on the death of the leucocytes, or rather phagocytes (" phago- lysis"), and that they do not exist in the body except under abnormal conditions. In any case when present they will probably prepare the bacteria for ingestion by the phagocytes, and thus are related to, if not identical with the Opsonins " (feast-preparers). The cytases or alex- ines are of proteid nature and are very unstable. The withdrawal of the salts from the serum by dialysis sus- pends their activity, which is restored on again adding them. This fact is evidently related to the lessened amount of chlorides excreted in the urine in all acute febrile processes, and especially in lobar pneumonia. The cytases or alexines may be regarded as an appliance common to every animal organism, for the dissolution of organized substances, whether bacteria, foreign red corpuscles, or other foreign bodies. They are the " Complements" of Ehrlich's classification. Acquired Immunity. — The immunity that is called natural is of a general kind, being a natural resistance to disease or bacteria of all kinds. That type of immunity called acquired, is, on the other hand, "specific" in kind, that is, an immunity from a definite or specific disease or infection. For this reason it is by some called specific immunity. (a). Acquired by an attack of a Specific Disease. — The fact that an attack of small-pox followed by recovery protected the individual from further attack, was a notable one during the epidemic prevalence of that disease. Similar 184 PUBLIC HEALTH BACTERIOLOGY protection was seen in regard to other eruptive and non-eruptive fevers such as scarlatina, measles, typhus, typhoid, and whooping-cough. On the contrary, some specific diseases do not protect, but one attack seems to render the individual more liable to another. Such are diphtheria, pneumonia, influenza, gonorrhoea, erysipelas, relapsing fever, and rheumatic fever. In all of these it is probable that some degree of immunity results, but is of very short duration, as has been definitely observed in cholera and some other diseases. In all cases, however short the immunity, it is absolutely specific against a certain infective agent or its poison, and is not due to a general increase of resistance. In fact the reduction of the general resistance following the specific infection is such as in some cases to predispose to other infections. Thus, tuberculosis not infrequently follows a severe attack of measles, whooping-cough, or typhoid. Recovery from an acute infective disease is due to a process of immunization going on during the progress *of the disease, which at a certain point or stage is able to prevent the further action of the infecting agent. The substances formed do not always exterminate the virus from the mucous surfaces ; and this is seen specially in typhoid fever, where the recovered patient may continue to excrete the living virus by the bowel or urinary discharges, and in diphtheria, where the virus may persist in the throat. Artificial Immunity. — Under this head may be classed together forms (b) and (c) of acquired immunity. (b). By Active Immunization, or Protective Inoculation— where the specific protective substances have to be formed in the body itself, as opposed to immunization by trans- ference of protective substances formed by active immunization in another animal, and called passive immunization. In active immunization, the individual or animal must undergo an infection followed by a reaction. By this means the protective substances are formed, and so the immunity is obtained only after the lapse of a period of time, when the immunizing apparatus of the organism is able to produce the protective substances in sufficient amount. The immunity thus evoked is of a more persistent type than that obtained by simply transferring IMMUNITY AND ANAPHYLAXIS 185 the protective substances from an immunized animal, because, in the first case, an immunizing apparatus has been set up which is able to produce an (apparently) indefinite amount of protective substances and over a long period, whereas in the second case, a definite amount of the protective substances is injected, and when this amount is used up the protection is at an end. In active immunization, we must distinguish a " specific immunity to bacteria," and a " specific immunity to their toxins," just as we have a natural resistance or immunity to bacteria, which is different from the natural resistance or immunity to certain poisons. Thus the immunity after diphtheria is mainly to the toxins (antitoxic). On the other hand, the immunity purchased by the injection of cholera vibrios, is merely to the bacteria and not to their endotoxins. Hence the injection of cholera spirilla into an animal previously immunized to the same, is followed by the death and dissolution of the spirilla, but if the dose is large enough, also by a fatal intoxication of the animal by the cell poisons thus suddenly set free. This is the chief cause of failure of immunization to those bacteria which do not act (as diphtheria and tetanus do) through soluble toxins, diffused into the blood stream. The immunization would require, in such cases, to be of a double nature, namely, antibacterial and antitoxic. Apparently, as usually induced, the former mainly, if not entirely, results. The methods of active immunization are based on the work and discoveries of Pasteur and his associates, and may be summarized thus : — (i) With living bacteria, virulent or attenuated. (2) With dead bacteria. (3) With the bacterial cell substances. (4) With soluble toxins or filtrates. (5) By feeding with toxic substances. 1. With Living Bacteria or Virus, Virulent or Attenuated. — (a). With virulent virus. Though the virus of small-pox is still unknown with certainty, inoculation of the small- pox, as introduced into England in 171 8 by Lady Mary Wortley Montagu (see her " Letters "), may be given as an example. The inoculated disease was usually mild in type, 186 PUBLIC HEALTH BACTERIOLOGY but at times was severe and even fatal. In contagious pleuro-pneumonia of cattle, subcutaneous injection of the lymph of a newly killed animal, into the tail, has proved protective. The animal sometimes loses its tail in part, the brunt of the infection apparently remaining localized. (b). With attenuated virus. This is accomplished in one or more of the following ways :— By cultivation of the organism in oxygen or a current of air, as first discovered by Pasteur in the case of the bacilli of chicken cholera. The attenuated bacilli, on injection, produced a non-fatal disease, which immunized the fowl, especially on repetition. By cultivation of the bacteria at high temperatures, e.g., anthrax bacilli at 420 to 430 C. By passage through a less susceptible species. This is the presently accepted explanation of vaccination for small-pox. Used by Pasteur for swine erysipelas, the bacillus of which is lessened in virulence by repeated passage through rabbits, but increased by passage through pigeons. Two inoculations were given, the first of the attenuated bacilli from rabbits, the second of the exalted bacilli from pigeons. By drying the virus, as in hydrophobia, where Pasteur found that the virus was exalted in virulence by successive subdural passage through rabbits, but diminished by passage through apes. He tried the immunization of dogs by the use of the diminished and increased viruses, but the results were too variable, so he tried drying the spinal cord of a rabbit dead of the disease. A cord thus dried at 220 C. over KOH (to absorb C02) for 1 to 4 days, still causes rabies in 7 days (the incubation period shortened from 14 days by the exaltation of the virus) ; but if kept longer, the incubation period is prolonged, until one kept 12 to 14 days has become inactive. The treatment consists in beginning with injection of an emulsion of this cord, and daily repeating with an emulsion of a less dried cord until within 15 days in mild cases and 21 days in severe, the strength arrived at is a 3-day-dried cord. Complete immunity thus takes 3 to 4 weeks, and so in cases coming under treatment at a late period, the method is condensed. Hoegyes uses fresh cord emulsified IMMUNITY AND ANAPHYLAXIS 187 in salt solution, of which dilutions are made, and injections made in reverse order of dilutions. Fewer accompanying symptoms (as erythema at the point of injection, backache, muscular pains, and occasionally temporary paralysis) are noted, and this result is attributed to the less amount of nerve tissue injected. By cultivation in a medium containing antiseptics in a dilute state ; e.g., in presence of carbolic acid 1-600, or potassium bichromate 1-5000, or sulphuric acid 1-200. By addition of weak antiseptic solutions to virulent broth-cultures preparatory to their injection, as advised by Behring for immunization of horses to diphtheria and tetanus. The antiseptic advised is iodine terchloride, ICI3, in strengths varying from 0*05 per cent to 0-4 per cent. Lugol's solution of iodine is also used. 2. With Dead Bacteria. — This method is simpler and safer, and in many cases confers the same degree of immunity, which is chiefly antibacterial. It is also used preliminarily to injection of living cultures, in the active immunization of animals. In the human being, it is used for cholera, plague, typhoid, and in the treat- ment by " vaccines " generally, as for staphylococcus infection, etc. 3. With Bacterial Cell Substances. — This method is a modification of that with dead bacteria. Instead of injecting the culture heated to 650 C, or some such tempera- ture, to kill the bacteria, the culture is subjected to various processes as in the preparation of Koch's original tuberculin. This is not purely bacterial cell substances, but is inter- mediate between the simply heated culture and tuberculin- R, which is an emulsion of the bodies of bacilli from which all the soluble substances have been extracted by grinding and treatment with distilled water (tuberculin-O). Hahn, following Buchner, has used mechanical pulverization of bacilli mixed with infusorial earth and quartz sand, and subjected to 300 to 500 atmospheres' pressure by hydraulic means, and has so obtained what he calls the cell-juices or bacterial plasmins, which he has used for immunization. " Cholera plasmin " and " typhoid plasmin " have both proved effective in immunizing guinea-pigs against intra- peritoneal infection with ten times the fatal dose of virulent 188 PUBLIC HEALTH BACTERIOLOGY bacteria. " Tuber culo-plasmin " after filtration is a clear pale yellow fluid, containing nucleo-albumin, which keeps indefinitely on addition of 20 per cent of glycerin and 5 per cent of NaCl. This preparation has been used with favourable results in guinea-pig tuberculosis. The reaction which follows the injection of a dead culture (local pain and swelling, rigor, depression, and anorexia) is not peculiar to any one bacterium, but follows upon the subcutaneous injection of all bacterial emulsions, and even of innocuous and living bacteria (Buchner, 1890). 4. With Soluble Toxins or Filtrates. — This method was first successfully used by Salmon and Smith, who showed that pigeons could be rendered immune to hog cholera by treatment with filtrates of hog-cholera bacilli (1886). It is now used for the immunization of horses to diphtheria and tetanus toxins, the immunity being afterwards heightened by the injection of virulent cultures, if Behring's advice is adopted. This use followed on the observations of Roux and Yersin, who showed in 1886, as a " result of splendid research " (Buchner) that the poison of diphtheria is extremely susceptible to heat (being destroyed at 650 C), and is carried down mechanically by chemical precipitates such as calcium phosphates, properties which until then had been recognized mainly in the digestive ferments or enzymes. Brieger and Fraenkel confirmed these results, and showed that the poisons or toxins of diphtheria and tetanus can be obtained in a moderately pure form by precipitation with absolute alcohol. These poisons gave the reactions of albuminous substances, and were hence at first called " toxalbumins." As they are now believed to be non-proteid, the name " specific toxins " is to be preferred. A specific toxin is one which, on injection, causes all the symptoms of the infection in question. 5. Active Immunization by Feeding has been successfully used by Ehrlich for the poisons ricin and abrin, and with less success by Fraser against snake venom. In bacterial infections it has proved, so far, tedious, and the immuni- zation slight in amount. (c). By Passive Immunization. — Behring in 1890 IMMUNITY AND ANAPHYLAXIS 189 discovered that the blood serum of an animal actively immunized against diphtheria, when injected into another animal, is capable of rendering the latter insusceptible to what would otherwise be a fatal dose of diphtheria toxin. That is, the serum was able to destroy the diphtheria poison. In conjunction with Kitasato he afterwards proved the same for the tetanus toxin. The transference of the immune serum, in both cases, protects against the specific poison, and so allows the natural resistance of the body to overcome the bacilli. As the new animal body has thus supplied to it an antidote to the microbic toxins which have been shown to produce the symptoms of the disease, no reaction to these toxins occurs (where they have been completely and early destroyed), and so no active immunization occurs. The immunity conferred is, therefore, of a transient nature, and lasts only as long as some of the immune body in excess persists in the blood. Any such excess is destroyed or excreted within eight to fourteen days, and so the immunity may be expected to be absent thereafter. These remarks apply to " antitoxic sera," like those obtained in diphtheria and tetanus. The other form of passive immunization by " antibacterial " or " antimicrobic " sera, has proved unsatisfactory in use, for the reasons given on page 185. The action of the latter sera is not so simple as that of antitoxic sera, but is due to one or more of the following factors : (1) Bactericidal or lysogenic action, that is, death or solution of the micro- organisms ; (2) Opsonic action, or the rendering the bacteria more susceptible to the phagocytic action of the leucocytes ; (3) Agglutinative and precipitative actions, that is, clumping of the bacteria, or precipitation of their soluble products. Antitoxic Sera. — The actual mode of manufacture may be conveniently given here. Diphtheria antitoxin may be taken as the type. A culture of B. diphtherial in meat-infusion broth (containing 1 to 2 per cent of added peptones, and after being made neutral to litmus, having 7 c.c. of N/i NaOH added per litre) is incubated for three weeks at 370 C. A strongly toxic fluid is thus produced, which is filtered through a Chamberland candle into a sterile flask, care being taken to avoid exposure to bright light. 190 PUBLIC HEALTH BACTERIOLOGY At the first attempts at immunization, the animals died of chronic poisoning. To avoid this, it is now usual to begin with very small doses of the toxin weakened by the addition of iodine terchloride or Lugol's solution (used in Gram's method of staining, I in KI in water). A young vigorous healthy horse (4 to 6 years old) is chosen, and 0-5 c.c. of toxin mixed with an equal quantity of Lugol's solution is injected subcutaneously in front of the shoulder- blade, using a large needle connected with a syringe by a piece of rubber tubing. (The skin is previously shaved and sterilized.) After the reaction has subsided, usually in 5 to 8 days, a few days' interval is given, and the next dose is administered, either 1 c.c. -j- 1 c.c. Lugol's solution or 05 c.c. pure toxin. The amount given is thus gradually increased by \ c.c. until finally in three to four months the antitoxic value of the animal's serum is such that a dose of 300 c.c. of active toxin may be borne. The immunizing process must not be pushed too rapidly, other- wise the health of the animal will suffer. Serum is then obtained by inserting a sterile needle into the jugular vein of the horse, and collecting the quantity of blood desired (up to 6 litres at a time) through a rubber tube into sterile Erlenmeyer flasks containing solution of citrate of soda. These are allowed to stand until the corpuscles settle, and the plasma is poured off into another flask, and allowed to clot. Separate the serum, and filter. If filtered at once, it is found to precipitate again on standing, hence it is better to allow it to stand a few days before filtration. Bottle or tube, after adding 05 per cent carbolic, but before this it should be standardized. Standardization of antitoxic serum is very important, so that accurate dosage may be determined, and also so that one strength may be aimed at, since in the making it is liable to variation. The making of such a standard is not an easy task, because no two samples of toxin are of exactly the same strength, nor are even two samples of the same toxin tested at different times. This is another way of saying that toxin is a very variable substance, but fortunately antitoxin is not so variable. Hence Ehrlich chose as his " immunity unit," the amount of antitoxic serum which will neutralize 100 times the minimum lethal IMMUNITY AND ANAPHYLAXIS 191 dose (M.L.D.) of toxin for one guinea-pig of 250 grm. weight and which kills it within 5 days ; the serum and toxin being mixed together and made up to 4 c.c. bulk, and injected subcutaneously, the animal survives the time- limit. A serum containing one such unit in 1 c.c. is called "normal serum" or "normal diphtheria antitoxin" (D.A.N.), while a serum 1 c.c. = 1,000 M.L.D. is spoken of as ten times normal (D.A.N.)10, etc. One c.c. of the normal serum is said to contain one " immunization unit." Work- ing back from quantities of serum of known strength (anti- toxic) and preserved in a dried state in a vacuum and in a dark cool place, the potency of any toxin at hand can be determined, and against this latter, any newly prepared serum can be standardized. In this way a fairly uniform standard can be maintained. The usual antitoxic serum on the market contains 2000 " immunity units " or shortly, units in 4 to 5 c.c. of serum and equal to 200,000 M.L.D. High-potency sera are prepared so that 5 c.c. contain 5000 units, and correspondingly. The serum keeps very well for at least one year in a cool dark place. The durability of the serum is tested by keeping back some of the bottles, and from time to time examining their activity, and if it is found to rapidly diminish, all the bottles bearing the same number are recalled. OTHER IMMUNITY PHENOMENA. Pfeiffer's Phenomenon. — In 1894, Pfeiffer showed that when cholera spirilla are injected into the peritoneal cavity of cholera-immune guinea-pigs, the spirilla rapidly swell up, become granular, and often undergo complete solution. The same result could be observed in a normal animal, if a protective amount of cholera-immune serum were injected at the same time. The constituents of the blood serum which cause this result are spoken of as " Bacteriolysins." It was soon shown that the same result followed the mixing of the spirilla and the serum in a test tube under suitable conditions. The same phenomenon was thereafter observed for other micro-organisms. Agglutination. — In 1896, Grueber and Durham, investigating Pfeiffer's phenomenon, found that when a 192 PUBLIC HEALTH BACTERIOLOGY quantity of immune serum is added to a broth culture of the respective bacterium, flake-like clumps sink to the bottom of the tube, and the supernatant liquid becomes clear. Grueber also showed that the immune serum would affect in the same way, though less powerfully, closely alhed bacteria. The substances causing this are called " Agglutinins," and were thought to be the same as the " immune-body M concerned in Pfeiffer's reaction. Both are comparatively thermostabile, but the agglutinins cannot be reactivated by the subsequent addition of normal serum. They do not act if NaCl is absent. Precipitation. — In 1897, Kraus showed that precipi- tates were formed when filtrates of cultures were mixed with the corresponding immune serum. The " Precipitins," like the agglutinins, are inactivated by heat (6o° to 700 C.) and cannot be reactivated (see Serum Precipitation, below). Haemolysis. — Bordet, in 1898, showed that the serum of an animal which has been repeatedly injected with the red corpuscles of another, acquires the power of dissolving the red cells of that other, and that this power is lost on heating to 550 C. but is regained by the addition of serum of a non-treated animal. Other " cytotoxins " (cell- destroying antibodies) similarly produced are : leucotoxin, nephrotoxin, spermato toxin, hepatotoxin, pancreatoxin, suprarenal toxin, etc. Serum Precipitation. — Like haemolysis, this subject is closely allied to the reactions induced by bacteria. When the serum of one animal is injected repeatedly into another animal of a different species, a substance forms in the first animal's serum, which causes, in a mixture of the two sera, a cloudiness or precipitate to form. This substance is called " precipitin," and is specific for each species, in high dilutions, as in the case of the other reactions. The precipitins, whether formed from bacterial or serum stimulation (or casein of milk, etc.), are all inactivated by heating to 6o° to 700 C, but can not be reactivated by the addition of normal serum or any known method. Such inactivated serum, however, if mixed with a certain amount of active serum, is able to prevent IMMUNITY AND ANAPHYLAXIS 193 the latter giving a precipitate. The precipitins are there- fore conceived to be built up of two atom groups, one thermolabile and the other thermostabile. Unlike agglutinins, they have not been, so far, shown to exist in normal serum. This reaction is used in forensic work, to determine the character of blood-stains, whether human or not. Opsonic Action. — In 1903-4, Sir Almroth Wright undertook a systematic study of the phenomenon of phagocytosis, and showed that phagocytosis depended on a substance present in the serum, which acts on the bacteria (and not on the leucocytes), becomes fixed to them, and makes them a prey to the leucocytes. To this substance he gave the name " opsonin " (feast preparer). This substance is present in normal serum, but can be increased by immunization. It is destroyed by heating to 55° C. Leucocytes washed with salt solution have no phagocytic action. On the other hand, if the bacteria are exposed to the action of serum, and then washed free of it, they can be phagocytozed by the washed leucocytes. It is hence inferred that the opsonin becomes bound to the bacterium. A similar substance has been described in immune sera after heating (bacteriotropin), but it is specific for the corresponding bacterium, while the opsonin in normal serum is non-specific. There are thus two opsonic substances ; that present in normal serum, which is thermolabile, and that present in immune sera, which is thermostabile. In opsonic estimation, both factors are at work where the person is being gradually immunized to a particular bacterium. The whole question is still complex and full of difficulties, and requires further elucidation. Technique of Opsonic Estimation. — The method of Leishman is very simple. Take a capillary pipette, fitted with a rubber nipple. Make a mark on the stem ; draw up fresh blood from the finger to the mark. Then draw up a little air to make an air-bubble, which separates the blood from the bacterial emulsion now drawn up to the same mark. The two fluids are then mixed by being blown out on a glass slide, and drawn back repeatedly. Finally, the drop is placed on the slide, covered with a 13 194 PUBLIC HEALTH BACTERIOLOGY cover -slip, and incubated at 370 C. for 15 minutes. Thereafter a film is made and stained by Leishman's method, and the number of bacteria present in 50 poly- morphonuclear leucocytes is observed. This number divided by 50 gives the " opsonic index." Wright's method is more elaborate and specific. He uses (1) Leucocytes from the observer's blood repeatedly washed with saline solution (o-85 per cent) ; (2) Bacterial emulsion in salt solution ; (3) Serum from the blood to be tested, free of clot and cells. These are now mixed, as above, in equal amounts, in a capillary pipette, and the mixing is made thorough by blowing out and sucking in, for ten times. The mixture is then drawn into the tube of the pipette, the end sealed, the rubber nipple removed, and the tube put into the incubator for 15 minutes. The tube is then removed, the end broken off, the contents are again mixed, and films made, dried or fixed, and stained as desired (Jenner, Leishman, or Giemsa), and bacteria counted in 50 to 100 cells. A control is done with normal serum, and the "Opsonic Index" taken is the quotient of that got with the patient's serum divided by the index got with the normal serum. The latter, to minimize variation, may be made by mixing the serum of several healthy individuals. The number of bacteria per leucocyte (polymorphonuclear), in any one estimation, is also spoken of as the " Phagocytic Index," and the opsonic index is thus the proportion between the phagocytic index for the patient's serum and that for the normal serum. A modification of the method is to take a number of dilutions of the patient's serum and of the normal serum and to estimate in all these not the phagocytic index, but the percentage of leucocytes which act as phagocytes, i.e., the "Percentage Index," and the indices of the corres- ponding dilutions can be compared. The bacterial emulsion used is likewise thinner than in Wright's method. By the same process, the dilution of the serum at which phagocytosis is absent or very slight, can be determined. This is called the " Opsonic Coefficient of Extinction." Wright's Vaccine Therapy consists in injecting killed bacterial cells into the infected individual, in order to raise the phagocytic index to that particular cell. He IMMUNITY AND ANAPHYLAXIS 195 began with chronic staphylococcal infections, and in such cases good results were obtained. The method has been applied to infections by tubercle bacilli, streptococci, gonococci, pneumococci, and other bacteria. If possible, a culture is made from the actual bacteria causing the infection. From the culture so made, an emulsion in sterile salt solution is obtained, and the emulsion sterilized at the lowest possible temperature (usually 65 ° C. for one hour), and an agar inoculation made from the presumably sterile emulsion, and incubated for twenty-four hours to see if really sterile. The bacterial content of the emulsion must be estimated, so as to be able to know how many are being injected, and to inject a definite quantity. This is done by mixing equal quantities of the emulsion (whole or diluted) and fresh blood, and making films, and staining. The number of bacteria to red cells is noted over a field made by drawing a circle with a blue pencil on the lens of the eye-piece of the microscope. The number of red cells in the blood being known (say 5 million per c.mm.), and the relative proportion of bacteria in undiluted emulsion to the red cells being, say, 700 to 400, then as 400 : 700 : : 5,000,000 : x = 8,750,000 bacteria per c.mm. If the emulsion had been diluted, then the result would have to be multiplied by the number of the dilutions. It is preferred that the content be estimated before sterilization, as some of the bacteria may undergo disintegration during that process. Where the blood serum has an agglutinating effect, the red cells are separated and mixed with salt solution. It is better to render motile bacilli still by having a little formol in the saline solution. From the stock emulsion thus standardized and sterilized, appropriate doses are made by dilution with 0-5 per cent phenol or lysol solution, and put in glass bulbs, with capillary ends which are sealed. The ordinary dosage is to begin with 100 million and to repeat, if necessary using a larger dose, after estimating the opsonic index, and only if this is rising. Numerous observations after the injection of such vaccines have shown that the opsonic indexi^falls for some time thereafter (" Negative Phase ") and then begins to rise, and usually ascends to a higher level than before. Another injection during the negative 196 PUBLIC HEALTH BACTERIOLOGY phase tends to accentuate it, while after the increasing or " Positive Phase " has begun, an injection causes it to reach a still higher level. The negative phase is usually completed in twenty-four hours, and the positive in three to four days. Wright recommends that the succeeding injection should be given when the positive phase has just reached its summit. In tuberculosis, Koch's bacillary emulsion is used, and the dose is minute, ^qtj- mgr-> gradually increased to TTrVr7 mgr- In vaccination against, and in, enteric fever, a standardized strain of Bacillus typhosus is used. Leucocyte Extract. — The action of the leucocytes in phagocytosis, and of the alexines, which some believe to be derived from them, led Hiss to experiment with leuco- cytic extracts. These were obtained by the intrapleural injection of aleuronat, which produced a copious cellular exudate in 24 hours. The animal being used (a rabbit) is killed, and the exudate removed, with every precaution to ensure avoidance of contamination, and the cells are obtained by centrifugalization. The deposited cells are treated with sterile distilled water, and thoroughly beaten with a platinum spatula. Smears are made, stained by Jenner's method, and examined for bacterial contamination ; cultures are made to detect the same ; more sterile water is added, and the steps are repeated after incubating for 8 hours. If no bacteria are found, the resulting fluid is put into the refrigerator until used. Such extracts of exudate cells, on intraperitoneal or subcutaneous injection, have markedly modified the course of infections in the rabbit and guinea-pig, prolonging life, and in some cases prevent- ing a fatal issue from an otherwise lethal dose. Beneficial effects have been observed in man in lobar pneumonia, erysipelas, and in staphylococcal infections. The action of the extract on the bacterial products or toxins seems to be a neutralizing or destroying one. The substances present in these extracts have been called " endolysins," and are different from the serum bacteriolysins, (1) in not being inactivated under 8o° C. ; (2) when heated above 8o° C. they are destroyed, and cannot be reactivated by the addition either of fresh serum or of unheated leucocyte extract. They are not increased by immuniza- IMMUNITY AND ANAPHYLAXIS 197 tion, each leucocyte probably having a definite quantity within its substance. Aggressins. — The great susceptibility of some species of animals to infection by certain bacteria, while their serum nevertheless possessed marked bactericidal power against these bacteria, suggested to Bail the theory that these bacteria secrete definite substances, which protect them against phagocytosis. Such substances he called " aggressins," and they are therefore antagonistic to the opsonins. They are probably not produced in test tube, or only to a slight degree. He based this theory on two observations, namely, that sub-lethal doses of bacteria, injected along with a small quantity of " aggressins" were rapidly fatal ; and that animals could be immunized against the corresponding bacteria by the injection of the aggressins. The aggressins were got, as detailed on page 177, in the exudate into a serous cavity of an animal killed by the injection of a dose of a particular bacterium into the serous cavity, and from which exudate the bacteria are carefully removed. This theory has been attacked on the ground that the aggressins are merely the bacterial toxins, probably endotoxins, liberated in the living body. The fact that such exudates are usually cellular, along with what has been said above of the action of leucocyte extract in some infections, tends to confirm this criticism. In fact, it may be put this way : When there is a high natural resistance to a bacterium, the alexines and opsonins are able to overpower it in all average infections and prepare it for phagocytosis and subsequent destruction. On the other hand, where the natural resistance is low, these agents do not succeed in preventing the growth of the bacterium, which in its growth elaborates various substances, some of which reduce the resistance still further, and so progres- sive infection results. If this infection is not too severe, the immunizing apparatus throughout the body, stimulated by the diluted toxins (extra- or infra-cellular) present in the blood stream, produces an excess of antibodies and anticells (phagocytes), and in this way may attain the objective of active immunization. If the infection is too acute, paralysis of the immunizing apparatus is the result. 198 PUBLIC HEALTH BACTERIOLOGY THEORIES OF IMMUNITY. The rational explanation of all the phenomena of immunity which are known, is a task yet to be accomplished, It is perhaps better to have a working hypothesis only, as our ideas are being continually enlarged and modified. The many elaborate and complex experiments which have been performed and repeated by many observers are attended by so many consenting circumstances, of some of which we are totally ignorant, that it is not surprising that the inferences from the same experiment are so varied and even at times so conflicting. The words of Pasteur, used in another regard, seem quite appropriate here : " In experimental science, it is always a mistake not to doubt, when facts do not compel affirmation. ... In my opinion the question is whole and untouched by decisive proofs." Any theory must take account of phagocytosis, the bactericidal power of normal serum, the results of immun- ization as seen in the formation of antitoxins, bacterio- lysins, agglutinins, precipitins, and opsonins, and any other phenomena which emerge in the further consideration of these. When the theory is built around the phagocyte, it is called a " cellular " theory ; if the body fluids are taken as the key, a " humoral " theory. The final explanation will probably lie in a judicious blending of these two theories. Metchnikoff's Phagocytic Theory. — In this theory immunization leads to a more rapid and greater leuco- cytosis in response to subsequent infection by the same agent. At the seat of invasion there is also emigration of the microphages from the blood-vessels into the tissues, or if in a serous cavity, the exodus is into the same, giving a cellular exudate. On examination of these cells, many of them are found (in bacterial infections) to contain bacteria in their substance. These are not simply dead bacteria, in process of removal, but living and virulent ones. At a later stage, the bacteria may be seen swollen, granular, and vacuolated, and finally disintegrated. On the other hand, the phagocyte may not be able to digest the engulfed bacteria, and may itself be killed ; and in that IMMUNITY AND ANAPHYLAXIS 199 case, the leucocyte itself will disintegrate. The substance present in normal blood, which is bactericidal, Metchnikoff calls " Cytase," and he holds that it is secreted by the phagocytes. The substance formed in immunization which, like the cytase, is bactericidal, he calls the " Fixateur," and also looks upon as a derivative of the leucocytes. He believes that these substances (at least, the cytase) are only set free in the blood-stream by the destruction of the phagocytes. The action of opsonins and leucocyte extract all tend to confirm the importance of phagocytosis, and the probability that these cells, retaining the characters of the amoeba, retain also its marvellous adaptability, which is not usually seen or expected of the fixed tissue cells, which are so very highly specialized as to function. Metchnikoff therefore believes that for every infection the leucocytes develop a power of resistance, which may be revived on any subsequent infection, and so protect to a greater or less degree. Ehrlich's Theory is more complex. The discovery of antitoxins led to explanations of their action. At first they were thought to destroy the toxin, but this simple explanation was set aside by the experiments of Calmette on snake poison, which is thermostabile up to ioo° C. He noted that non-toxic mixtures of the toxin and antitoxin became toxic again on heating, the inference being that the toxin was bound or inactivated by the antitoxin, which is destroyed on heating above 6o° C, and so the more stabile toxin is again left free. Further, C. J. Martin and Cherry demonstrated the close resemblance of the union to that of definite organic compounds, by an experiment in which they tried to pass toxin-antitoxin mixtures through a Chamberland bougie, the pores of which were filled with gelatin. In previous experiments they found that under 50 atmospheres of pressure, toxin passed through but antitoxin did not. In toxin-antitoxin mixtures, if filtered at once, all the toxin came through ; but after standing for variable periods, less came through the longer the time, until two hours after mixing, no toxin passed through the filter (or dialyser) . Then Ehrlich showed, using ricin and antiricin, that definite quantitative proportions of the toxin and antitoxin entered into the 200 PUBLIC HEALTH BACTERIOLOGY reaction. The standardization of diphtheria toxin and antitoxin was the next step. Von Behring called a toxin containing ioo minimum lethal doses (for a 250 grm. guinea- pig) in 1 c.c, a "normal toxin solution" (D.T.N^M250), and a serum capable of neutralizing it c.c. for c.c, a " normal antitoxin " or an " antitoxin unit." Ehrlich, in working at the subject, more exactly measured the toxin unit by introducing a time-limit, namely, that one unit must kill the guinea-pig in 4 to 5 days. He also varied von Behring's method of testing the antitoxin, by first mixing the toxin and antitoxin outside the body, and thereafter injecting ; whereas von Behring injected them separately and at different parts. He prepared in this way an antitoxin, which he kept in a stable condition by drying in a vacuum and preserving in the dark in a dry atmosphere and at a low temperature. With this antitoxin he is able to standardize new toxins, and from them new antitoxins. In the course of this work he made some discoveries. In the first place, while the death of a guinea-pig in 4 to 5 days gave a fair measure of 1 toxin unit, when 100 such units were mixed with the amount of antitoxin necessary to neutralize, namely, 1 antitoxin unit (which was determined from previous measurement), and injected into a guinea-pig, it was not easy to estimate whether there was exact neutralization, or less, or more. If the antitoxin were markedly insufficient to neutralize the toxin, then symptoms such as paralysis, etc., would arise which would proclaim this. But the conditions of the experiment were such that no marked signs could be expected. The further test, therefore, was devised, namely to find the amount of toxin which, plus 1 unit of antitoxin would still be able to kill a guinea- pig, on injection, in 4 to 5 days. (When a new serum is to be standardized, the amount of serum which, mixed with this last-mentioned amount of toxin, just suffices to prevent the death of the guinea-pig before 4 days, is taken as one unit of antitoxin.) Theoretically one might have expected that, if 1 toxin unit killed a 250 grm. guinea-pig in 4 to 5 days, and 1 antitoxin unit exactly neutralized 100 toxin units, the injection of 1 antitoxin unit mixed with 101 toxin units would have left 1 toxin unit free to have killed IMMUNITY AND ANAPHYLAXIS 201 the guinea-pig in 4 to 5 days. Piactically this was not found to be so, but that a considerable excess of toxin units is required to kill the guinea-pig, (stated by Ehrlich to be 100 toxin units, and by others as intermediate amounts). On this basis Ehrlich has built a whole structure of epitoxoids, toxoids, prototoxoids, syntoxoids, and toxons, designed to explain on the laws of chemical equivalence and differences in chemical affinity, the apparent contra- diction of these results. It is right to point out, however, that the basis is not a chemical one. The death of a guinea-pig in 4 to 5 days cannot be compared, though it follows on an injection of toxin, to the appearance, more or less immediately, of a precipitate in a liquid to which some other chemical body has been added. Also, the relationship between the mixture of toxin and antitoxin which just causes no symptoms, and that mixture which kills a guinea-pig in 4 to 5 days, is an arbitrary one, and any apparent numerical relationship should be regarded as fortuitous until other evidence shows that it is more than that. Again, the admitted instability of the toxins, and probably of antitoxins, even under every precaution, renders the results equivocal. In brief, Ehrlich's theory is that antitoxin has a valency of 200 for toxin, and that some of the bonds of antitoxin can be satisfied by degenera- tion products of toxin, prototoxoids, deuterotoxoids, tritotoxins, of alpha and beta varieties, and by toxons, which are not derived from toxin but are present in the toxin fluid at first. [" Valency = 200 " cannot be accepted in the chemical sense. It only means that, starting with an amount of antitoxin which neutralized 100 toxin units elsewise defined, it was found that when the conditions were changed, the antitoxin, in some way or other, neutralized 200 of these same toxin units, or in the experiment appeared to do so. If the amount of antitoxin used at first had been the amount required to neutralize 1 toxin unit (and von Behring advised the larger quantity only for safety), the amount found in the second experiment would have been 2 by inference. The mixture of the toxin and antitoxin before injection may be a factor of moment in regard to this change of valency.] Ehrlich calls the quantity of toxin which just neutralizes 202 PUBLIC HEALTH BACTERIOLOGY i antitoxin unit, " limes zero," expressed as L0 ; and the quantity required to neutralize I unit of antitoxin and yet, on injection, kill the guinea-pig in 4 to 5 days, " limes death, expressed as L +. Then, according to his theory, if T represent 1 toxin unit, Lo = iooxT and L+ = L0 + ioixT == 20ixT. Ehrlich's side-chain theory is based on his previous researches on the oxygen requirements of the organism, linked up with those on diphtheria antitoxin. Borrowing the language of organic chemistry, he likens the highly complex albuminous and other molecules of animal nutrition, to those complex compounds of the aromatic series which chemists have dissected into a central group, in which the elements may be represented as a hexagon or ring of the benzene type, and the various other parts as side-processes or side-chains. Thus benzene has derivatives like the following, the added groups of which may be spoken of as side-chains. .CH-CEL Benzene CH< >CH CH - CH' O.CH CH - C Acet. CH3.CH2.-C/ >C-CO.OCH Eugenol XCH - CHX Applying the same ideas to living cells, Ehrlich believes that these cells have side-chains which have certain special affinities. . In this way the diphtheria toxin may be supposed to be bound to certain nerve cells ; and likewise tetanus toxin. The side-chains he calls receptors. When thus bound by a toxin molecule, they are supposed to be useless to the cell and are cast off into the blood-stream, and the cell is supposed to be stimulated to produce more ; not only so, but stimulated to produce an overplus which is alleged to be then cast into the blood-stream, as the cell would become overstocked. Thus he accounts for the presence of antitoxin free in the blood, the free receptors acting as antitoxin to the toxin circulating. The toxin, which thus unites with the antitoxin, he conceives as IMMUNITY AND ANAPHYLAXIS 203 having two affinities : one which unites with the antitoxin, the haptophore ; the other, the toxophore, by which the harmful effects are produced. These two affinities are the affinities of two different atom-groups, of which the toxin is supposed to be composed. In the toxoid bodies,, the toxophore group is altered or wanting, but they can still bind antitoxin, in virtue of their haptophore group. For other forms of immunity, which are more complex than the toxin-antitoxin one, some elaboration is required. In natural immunity, the blood contains a thermolabile substance which is bactericidal. To this substance Buchner gave the name " Alexine " ; Metchnikoff spoke of it as " Cytase " ; and Ehrlich renamed it " Complement."" In active immunization, a more thermostabile substance,, bactericidal in nature, appears in the serum, and has been variously called "Fixateur" (Metchnikoff), "Substance Sensibilisatrice " (Bordet), "Immune Body or Ambo- ceptor " (Ehrlich). This substance is found to act only in the presence of complement, and hence Ehrlich' s conception that it has two combining affinities which must be satisfied to produce bacteriolysis. The one affinity binds it to complement, the other to the immunizing substance (bacterium or red blood cell, leucocytes and other body cells, toxins, ferments) ; called the Antibody - producer, or Antigen. The immune body he therefore called an amboceptor, or receptor with two hands, which he described as the cytophile haptophore and the comple- mentophile haptophore. He also believes that the complement is composed of two parts, a haptophore group and a zymophore group. According to Ehrlich, the complement is unable to act directly on the antigen or antibody-producer, but only when connected by the immune body or amboceptor. Bordet, however, believes that neither antigen nor immune body has any affinity for complement, until when they are united they can absorb the complement, but not through the immune body. The hypotheses of Ehrlich have been used to explain agglutination, precipitation, and other phenomena, with sundry modifications. It is at present unnecessary to follow the theory further, because in these fields its explanations have been most called in question. This is not to be 204 PUBLIC HEALTH BACTERIOLOGY wondered at considering the enormously increased and increasing complexity of the subject-matter. Under either theory of immunity, the immune-body produced in active immunization is specific, that is, there is a special immune-body produced for each antigen. On the other hand, the complement, or alexine, or cytase, is believed to be one and the same, though Ehrlich and his school have argued in favour of specific complements for specific amboceptors. FURTHER IMMUNITY PHENOMENA. These can be more easily followed after the terms used in the theories have been acquired. i. Filtration of Serum. — Muir and Browning found that on filtering serum through a Chamberland bougie, the immune-body passed through, and the complement did not. 2. Fixation of the Complement. — Bordet and Gengou planned an experiment, called the M Bordet and Gengou Reaction," to demonstrate the presence in a given serum of a specific immune-body, even in very small quantities. To this reaction the term " Fixation of the Comple- ment " is now commonly applied, and has its best known practical use in the " Wassermann Reaction." They performed two parallel experiments, (i) and (2), in which they used the following mixtures : — (1). Heated immune plague serum -f plague bacilli emulsion -f fresh normal serum. (2). Heated normal serum + plague bacilli emulsion -f- fresh normal serum. Set aside for five hours at blood heat. Then added to each mixture : (a) geated hemolytic serum + washed red blood cells. Obs*e?v^ results : Mixture (1) shows no haemolysis ; mixture (2) shows haemolysis. The explanation of this phenomenon is after this manner : Looking at (a) we see that the mixture there requires the addition of complement to produce haemolysis, since the complement in the serum has been destroyed by heat. Therefore, when mixture (2) produces haemolysis, we infer that it must have supplied complement ; and IMMUNITY AND ANAPHYLAXIS 205 similarly that mixture (i), since it did not produce haemo- lysis, could not have supplied any complement. We have therefore to examine mixtures (i) and (2) carefully to determine the cause of their different actions. To do this,, they may be rewritten thus : — (1). Specific immune-booly -f- specific antibody-producer -f- complement. (2) . Non - specific immune - body -f- specific antibody- producer -f- complement. They differ only in their immune-bodies (normal serum is believed to contain non-specific immune- bodies) . It is therefore inferred that in (1), since no complement is left free, it has become bound by the joint action of the specific immune-body and the corresponding antibody-producer. In (2), since the immune-body and the antibody-producer do not correspond, no binding or fixation of the complement occurs, and so haemolysis takes place on adding (a). It should be noted here, that the quantity of complement used is determined by previous experiment, as the presence of an excess over the quantity which can be bound by the amounts of immune-body and antibody-producer would lead to haemolysis, even in (1). This reaction is capable of many applications in the determination of specific immune-bodies in a serum and of specific antihoffo-prorhicers (antigens) in a serum. We shall here describe briefly the so-called " Wassermann test " for the diagnosis of syphilis, by determining the presence in the patient's blood of an immune body, capable when mixed with syphilitic antibody-producer (antigen) of causing fixation of complement. Wassermann Test. — Requisites : (1) Specific antibody-producer, referred to usually as the antigen. (2) Red blood cells of a sheep, washed free of com- plement. (3) Serum of a rabbit's blood, haemolytic to sheep's red cells, heated before use, to destroy its complement. (4) Fresh guinea-pig's serum, to supply complement. (5) Serum from the patient, heated to 560 C, which serum is to be tested for the presence of specific immune- body or antibody. 206 PUBLIC HEALTH BACTERIOLOGY i. The antigen at first used was a salt solution extract of the liver of a syphilitic foetus. Alcoholic extracts have also been used, and it has been discovered that the -extracts of normal liver and spleen, and even a I per cent emulsion of lecithin, will act as antigen. (This shows how little we must know of possible fallacies, in tests like this, with highly complex bodies like liver extract. It does not impugn its specificity for pure antigens, though it suggests the possibility of two different substances, with similar affinities for the immune-body produced by one of them. Of course the probability of this other substance being present, under the conditions of the experiment, is small, but must never be lost sight of.) The quantity of antigen to be used has to be carefully predetermined, as large excess of antigen has been found to cause binding of the complement, even in the absence of the immune-body. A series of trials of varying quantities of antigen with the same amount of complement in each case, shows the largest quantity of antigen which can be used without exerting this action. This amount of antigen is arranged, by proper dilution, to be present in i c.c. 2. Washed Red Blood Cells. — Some sheep's blood is gathered under aseptic precautions into a small sterile flask, containing sterile solution of sodium citrate (0-5 per cent) and sodium chloride (0-85 per cent). The corpuscles are separated by cehtrifugalizing, and washed repeatedly in the same way with sterile salt solution, to get rid of serum-complement and serum-precipitins. They are then brought down to a 5 per cent emulsion in salt solution, by mixing them with 19 times their bulk of the same. 3. Hemolytic Serum for Sheep's Cells. — This is obtained by injecting a rabbit with washed red blood cells of a sheep, obtained as above. Of the 5 per cent emulsion, three or four injections are given at intervals of 5 to 6 days ; the first injection of 5 c.c, the second of 10 c.c, the third of 15 c.c, and the fourth of 20 c.c. ; intravenously or intra- peritoneally. Ten days later than the final injection the serum is obtained by drawing blood from the carotid artery, allowing it to clot, and pipetting off the serum. A high-potency serum is desirable so that a small quantity only may be required. This obviates or reduces the risk IMMUNITY AND ANAPHYLAXIS 207 of precipitates, due to precipitins in the rabbit's serum for sheep's serum (from insufficient washing of the injected corpuscles), acting on any sheep's serum present, from insufficient washing of the corpuscles used in the test. The serum is inactivated by heating to 560 C. The quantity of haemolytic serum to be used must be accurately deter- mined,' in relation to a definite amount of complement. This is the more necessary since it has been shown that, (contrary to Ehrlich's commonly accepted conception that immune -body and complement react in definite combining proportions), the haemolytic immune-body and a complement react in inverse proportions ; that is, the more immune-body, the less complement is required. The bearing of this on the test is that a very small quantity of complement left over by the combination of syphilitic- immune-body and antigen might suffice, in presence of large excess of h hemolytic immune-body, to cause haemo- lysis ; thus giving a negative result in a positive case. The quantity is determined by putting up a number of mixtures, each containing o-i c.c. of fresh guinea-pig serum to give complement, and 1 c.c. of the 5 per cent emulsion of sheep's corpuscles. To each mixture inactivated haemolytic serum is added, in smaller and smaller quantities. The smallest quantity which gives complete haemolysis is taken as the unit amount to be used in the test. The haemolytic unit may therefore be defined as " the smallest quantity of inactivated haemolytic serum which, in the presence of a stated amount of com- plement, is able to cause complete haemolysis in 1 c.c. of a 5 per cent emulsion of washed red blood cells." 4. The Complement. — This is obtained by drawing blood from a guinea-pig, allowing it to clot, and pipetting off the serum, or separating by the centrifuge. Such serum, kept at a low temperature, preserves its complement in a fairly constant amount for three days. 5. The Serum to be tested is got from the patient under aseptic precautions, from the median basilic vein, from the finger, or from the ear. Three to four c.c. are with- drawn, as 1 c.c. of clear serum is required to go over the tests and controls. It is inactivated by heating to 560 C. in a water-bath for 20 minutes. Noguchi advises 208 PUBLIC HEALTH BACTERIOLOGY 540 C. Serum can also be obtained from cerebrospinal fluid, etc. Process. — (a). In a test tube, put the following : — o*i c.c. of complement (fresh guinea-pig serum). 0*2 c.c. of inactivated test serum (from patient). ro c.c. of standardized antigen (liver extract, etc.). Add salt solution to make up bulk to 3 c.c. ; shake thoroughly, and place for 1 hour in incubator at 37*5° C. (b). Now add, 1*0 c.c. of 5 per cent emulsion of sheep's red cells. 2#o units of haemolytic serum (determined as above) . Shake thoroughly, and incubate again at 37-5° C. for 1 to 2 hours. (c). Observe result. (1). No hcemolysis. Immune-body or anti- body is present ; test positive. (2). Complete hemolysis. Immune - body or antibody is absent ; test negative. Several controls should be put up at the same time to preclude error. Such are : a test with known normal serum ; tests with antigen and complement alone to see that antigen is not fixing complement ; test of haemolytic serum and cells and complement with and without antigen to see that haemolysis is actually possible, and tests with known syphilitic serum with and without antigen. In all these controls, except that with known syphilitic serum with antigen, complete haemolysis should occur. Noguchi has modified the test by using human red cells and an anti-human haemolytic serum. This simplifies the test in that the patient's red cells may be used in testing his own serum, and in that no antibody for his red cells exists in his own serum (human serum at times contains an antibody to sheep's cells). Anti-human haemolytic serum is prepared and standardized in a similar way to that described as used in preparing anti-sheep haemolytic serum. Human serum, when used to provide complement, is said to vary more in its content than guinea-pig serum, to absorb 10 times as much immune-body, and to be less sensitizing. IMMUNITY AND ANAPHYLAXIS 209 Fleming's modification is to use the hemolytic immune- body in human serum for sheep's corpuscles, and thus he can use the complement in the patient's blood. This simplifies the process. D'Este Emery uses human red cells, sensitized with heated immune serum from a rabbit which has been injected with human cells. , He advises only 5 minutes' incubation in a water-bath at 380 C, which very appre- ciably shortens the time taken for the test. Browning, Cruickshank, and McKenzie describe " a method of carrying out the reaction which is very reliable in practice, and depends on the fact that the amount of complement absorbed by a mixture of serum and lecithin is increased on the addition of cholesterin, if the serum is syphilitic ; but not, if the serum is normal. Accordingly, two series of tests are carried out simultaneously — in the one, complement is added to the mixture of serum and lecithin ; in the other, to the mixture of serum, lecithin, and cholesterin. If more complement is absorbed in the second series than in the first, then the reaction is positive." Value of the Test. — Like the agglutination or Widal test for enteric fever, the fixation of complement or Wassermann test for syphilis has its limitations. It is almost always negative in healthy subjects, but quite often gives a positive reaction in those suffering from leprosy, scarlet fever, jaundice, yaws, sleeping-sickness, and the acute stage of malaria. In the primary stage of syphilis, a negative result is usually got in the first fortnight. Thereafter one may expect a positive resjilt in 50 per cent of the cases, and its absence has some weight. In the secondary stage, in the presence of a suspected rash, a positive reaction is got in 50 to 70 per cent of the cases. In the tertiary stage, with progressive lesions present, the absence of the reaction is almost conclusive proof that another diagnosis than syphilis must be sought. In later stages, a positive reaction shows that the patient is not cured. Does a negative reaction show that he is. cured ? If a positive reaction was got previously, then it 14 210 PUBLIC HEALTH BACTERIOLOGY is likely, always excluding treatment with mercury, which inhibits the reaction. If, at any stage, the patient is under treatment with mercury, a negative reaction has no value for diagnosis. Stop treatment for one to two months, and test again. Salvarsan or " 606 " also inhibits the test. In a few cases of syphilis, no reaction is given at any time. In congenital syphilis, the reaction may persist through- out life, and be present even where there are no evidences of active pathological processes. The examination of the blood of the mother will usually give corroboration, and that of the father may, but not necessarily. In tabes dorsalis, general paralysis, and aneurysm, a large number of positive results have been obtained ; about 60 per cent in tabes, and 99 per cent in general paralysis. In the latter it is got in the cerebrospinal fluid. Porges- Meier Reaction. — Equal parts of clear blood serum and a 1 per cent emulsion of lecithin or other lipoid substance in carbolized salt solution, are mixed and allowed to stand at room temperature for 5 hours. Normal serum causes no precipitate, but syphilitic serum does. This test is not nearly so delicate macroscopically as Wassermann's, and hence, while much more simple, is not so readily interpreted by the naked eye. Jacobsthal has studied it microscopically, by the aid of the* ultra-microscope. He found that with normal serum, the particles of the lecithin emulsion appeared as isolated brilliant points, showing active Brownian movements. With a syphilitic serum, these brilliant points were seen to run together to form a large and brilliant mass. Intermediate reactions were noted, in which partial agglomeration occurred into small brilliant masses or brilliant chains. These phenomena were tested against the Wassermann reaction, and were found to run parallel to it. Determination of an Antigen by Complement- Fixation. — The same principles apply, but in this case the immune-body present in the serum is known, and a serum is tested which is supposed to contain the corres- ponding antigen It has been applied to test blood for IMMUNITY AND ANAPHYLAXIS 211 the antigen of B. typhosus, using highly potent anti- typhoid serum obtained from an immunized rabbit. Deviation of the Complement. — Neisser and Wechs- berg, experimenting with mixtures of specific inactivated immune sera, specific antigen (bacteria) and complement, found that, beginning with the complement in excess, more and more bacteria were destroyed as the amount of immune -body was increased, up to a maximum. Beyond this, increase of the amount of immune-body lessened bacteriolysis, and finally in great excess, seemed to stop it entirely. To this phenomenon they gave the name of " deviation of the complement," in accordance with their theory that free immune-body has a greater affinity for antigen than immune-body joined to complement ; and so in great excess of immune-body, the immune-body appropriates all the bacterial receptors, to the exclusion of the immune-body joined to complement ; hence the cessation of bacteriolysis. This explanation cannot now be accepted, and so the term " deviation of the comple- ment " is unfortunate and should be dropped. It is possible that the explanation of the phenomenon should be sought on physical lines, the excessive dilution of the mixture with serum containing immune-body reducing the chances of bacteriolysis by the complement in the same time-limit ; and it is possible the complement may be inhibited by other constituents of the serum added. Heterolysins, Isolysins, Autolysins. — A haemolysin produced in the blood of one animal by the injection of the red cells of another species, is called a " hetero- lysin." When the hemolysin is produced by injection of red cells of a member of its own species, it is called an " isolysin." Both of these have been produced. The production of a haemolysin in an animal by the injection of its own red cells, has not been accomplished. If such a haemolysin were produced, it would be called an " auto- lysis" The injection of isolysins produces " anti- isolysins," which like the heterolysins and isolysins are specific. The search for autolysins is clinically significant, as a possible theory for paroxysmal hemoglobinuria. 212 PUBLIC HEALTH BACTERIOLOGY ANAPHYLAXIS. I. General Principles. — As already defined, anaphylaxis is a supersensitiveness induced in man and animals by the injection of certain substances, mostly, so far as is known, of an albuminous nature. The ideas on this subject have grown around anomalous phenomena following on the injection of diph- theria toxin and antitoxin. Thus v. Behring and others noted that occasionally animals highly immune to the toxin showed excessive susceptibility to small doses of the toxin. Very soon after the introduction of the serum treatment of diphtheria in 1894, certain symptoms were observed to follow the injection of the serum, which, at first ascribed to the antitoxin contained in it, were finally found to. be due to the horse serum which carried the antitoxin. These symptoms were hence called " Serum Sickness " or " Serum Disease " and were mostly a rash of an erythematous nature, and fever. These come on in most cases about the ninth day, but vary from the third to the nineteenth. Fever is not always present, and the rash is quite often urticarial, and occasionally scarlatini- form or morbilliform, is local to the point of injection or becomes general, is fugitive or persistent. Articular pains may accompany the rash, and may be severe. The frequency of these symptoms varies ; from 30 to 55 per cent of the cases treated with serum are stated to show them in some degree or other. Some oedema is also noted by Currie, as accompanying the rash. The similarity of these symptoms to those of the specific infective diseases is striking, and led to the name " serum disease," which has therefore an incubation period (after subcutaneous injection) averaging nine days, and a duration of two days on the average. That these symptoms were due to the horse serum, and not to the antitoxin, has been proved by such symptoms following the injection of the normal serum of the horse. Another group of symptoms which on rare occasions followed the use of serum, was not at once recognized as due to it, but by the accumulation of cases and certain special features connected with them, the causal action of the serum was IMMUNITY AND ANAPHYLAXIS 213 obviously suggested, and experiments on animals proved the truth of the inference. These symptoms differed from those of serum disease in that they came on immedi- ately or within an hour after the injection, were very severe, and in some- cases ended fatally. The earliest recorded case is that of the son of Professor Langerhans, who was given a prophylactic dose of serum, took ill at once, and died shortly afterwards. The next recorded cases were three communicated by Goodall to the Anti- toxin Committee of the Clinical Society (of which Committee he was a member). The first of these was that of a girl, aged 4 years, admitted to hospital suffering from diphtheria. She was given 4000 units on October 24th, 1897, and the same amount on October 25th and 26th. On Nov. 3rd there was a slight urticarial rash. On Nov. 30th she had a well-marked relapse of diphtheria, and was in j ect ed with 4000 units of antitoxin . ' ' Within twenty minutes of the injection, she was seized with shiverings, quickly followed by two convulsions. Seen a few minutes later by the assistant medical officer, the convulsions had' ceased, but the child was in a drowsy state, and the temperature had risen to 1050 F. . . . There was no rash. . . . During the night the child vomited several times ; about 6 a.m. on Dec. 1st a rash was noticed, and was a multiform erythema. It persisted till Dec. 5th, and while it was present the temperature remained up and the pulse was very rapid. On Dec. 5th and 10th there were twitchings of the mouth, and throughout the child was drowsy and apathetic, and had a bad colour. She slowly recovered and left the hospital well on Feb. 3rd." Similar cases could be multiplied, and some given in which the immediate reaction is limited to a local or general rash. Such cases giving an immediate reaction fall into two groups : (1) Where the serum has not previously been injected ; and (2) Where a dose of horse serum has been administered on a previous occasion (excluding doses given within the incubation period of the serum disease). 1. In the first group numerous fatalities have been recorded. In most of these, a history of asthma, or some similar condition, has been noted, and this is very important, since the subcutaneous injection of diphtheria 214 PUBLIC HEALTH BACTERIOLOGY antitoxin has been recommended as a cure for asthma. A record of a fatal result in a man of 52 years, who died in tonic spasm ten minutes after receiving the serum, elicited particulars of 16 similar cases, and in the " Therapeutic Gazette " for March 15th, 1909, a short account is given of these, with 14 others, making 30 in all, in which alarming symptoms followed shortly after the injection, and ended fatally in 16 cases. In 22 of the 30 cases there was a history of asthma or some similar affection. (Goodall in Public Health, January, 191 1). 2. Where there is a record of a previous administration of serum, while the immediate symptoms may be very alarming and dangerous, so far no fatality has been reported. The use of serum day after day in a severe case of diphtheria is not followed by these manifestations, unless a period of at least ten days separates the first dose from that causing the symptoms. It is this feature which suggested that the symptoms were due to supersensitive- ness to serum, following on an attack of serum disease. The condition, beginning ten days after the first injection of serum, has been present in persons after five years, and may yet be found at a longer period. Experimental work on the subject has shown that rabbits injected with horse serum are very sensitive to a subsequent dose, show severe symptoms, and often die. This has been called the " Phenomenon of Arthus." The " Phenomenon of Theobald Smith " is that guinea-pigs used to standardize antitoxin, when injected with a toxin-antitoxin mixture were always killed on the subsequent injection, after ten days, of normal horse serum. Otto, Rosenau, and Anderson, working independently, showed conclusively that the action of the serum was without relation to its antitoxin content ; that sensitation of the guinea-pig was most marked after 10 days ; that very small doses were efficient (o-ooi c.c. or less) ; that the condition was transmissible from mother to offspring ; that it was specific for the particular serum used ; that it was not a hemolytic nor precipitin action ; that the condition could be conferred on another animal by injecting it with the serum of a sensitized animal ; that a considerable dose of serum (5 c.c.) is required for the second injection ; and that the symptoms are prompter in IMMUNITY AND ANAPHYLAXIS 215 appearing if the second injection is made intraperitoneally, cardially, or cerebral ly, than if given subcutaneously. Sensitized animals which recover from the second injec- tion are thereafter immune (Antianaphylaxis). This im- munity may also be purchased by the injection of large quantities of the sensitizing serum towards the middle or end of the incubation period, but the duration of this immunity is believed by Otto to be short. In a number of cases where a second injection is given after an interval, no immediate reaction (within 24 hours) follows, but an "Accelerated Reaction," that is, the local and general symptoms of serum disease are noted earlier than in the previous attack, or than is the average where no record exists. The simplest explanation of the phenomena of anaphy- laxis is that of Wolff-Eisner, who holds that all proteid substances contain a toxic part, which does not produce an antibody when injected into animals. On the first injection a lysin is formed which breaks up the proteid, liberating the toxic part. A second injection results in the rapid liberation of the toxic part by the action of the already-present lysin, and hence the toxaemia. The profound affection of the nervous system, the general vaso-dilatation, and the more rapid action on intracranial injection, all suggest some substance which acts as a toxin on the nerve tissues. In this connection the use of serum for the cure of asthma is interesting, because it could be explained (if the patient survive) as diminished nerve- irritability to proteid. II. Anaphylaxis to White-of-Egg. — Besredka and Bronfenbrenner in their most recent memoire (Ann. de Vlnstitut Pasteur, Mai, 191 1), have studied very carefully the anaphylaxis produced by the injection subcutaneously of white-of-egg, both raw and heated to ioo° C. They produced active anaphylaxis by the injection of 0-5 c.c. of white-of-egg, diluted with an equal quantity of normal saline solution. The state of anaphylaxis appeared in 16 to 20 days ; with a smaller dose (yj-g- c.c), it appeared in 12 days. The injection of white-of-egg heated to ioo° C. into other guinea-pigs, 216 PUBLIC HEALTH BACTERIOLOGY produced a state of milder anaphylaxis than the raw white- of-egg. They hence conclude that the constituent of the white-of-egg which causes sensitation, while attenuated by heat, is thermostabile. This active state of anaphylaxis lasts for several months. Passive anaphylaxis was produced within 24 hours after the injection into a guinea-pig of the serum of a guinea-pig which had been previously treated with white-of-egg. The injection thereafter into the jugular vein of the sensitized guinea-pig of -g^ to T^ c.c. of white-of-egg, produced the classical symptoms of anaphylaxis, with death in 1 to 3 minutes. On the other hand, when the serum and the white-of-egg were injected at the same time, one guinea-pig showed a marked dyspnoea and some malaise immediately after the injection, but quickly recovered ; another showed slight respiratory distress ; and a third showed no reaction. In all three the injection was into the jugular vein ; in the first two it consisted of 2 c.c. of anaphylactic serum mixed with T±-$ c.c. of white-of-egg ; in the third guinea- pig, it consisted of 1-5 c.c. of serum mixed with 1 c.c. of 10 per cent solution of white-of-egg, equal to -^ c.c. of white-of-egg. Passive anaphylaxis, thus quickly induced, disappears more quickly than the active form, namely in about a fortnight. The mixture of the anaphylactic serum of the rabbit sensitized to white-of-egg, with white- of-egg, produced a precipitate. This was collected and diluted in normal saline, and injected into the jugular vein of two fresh (non-sensitized) guinea-pigs, and produced no reaction.* Whether active or passive anaphylaxis was conferred, the injection of a minute dose intravenously or intra- cerebrally produced the classical symptoms and shock resulting in death, described in the anaphylaxis due to serum and milk. Injected intraperitoneally, it is rare to get grave symptoms ; subcutaneously, no anaphylactic symptoms have been noted. This is markedly different from serum anaphylaxis, where the subcutaneous route is as fatal as the others. From this Besredka and Bronfenbrenner deduce that the most important thing in the production of the anaphylactic shock is the rapidity with which the antibody in the blood comes into contact IMMUNITY AND ANAPHYLAXIS 217 with the antibody-producer or antigen. White-of-egg being a viscous liquid is slowly absorbed from the peri- toneum or subcutaneous spaces, and so the combination of the antibody and antigen has not the suddenness or instantaneousness which follows intravenous injection. (This deduction suggests that the antigen may act in a catalytic manner, causing an amount of chemical action out of proportion to its own mass injected. In such a case, the symptoms may be partly due to the accom- paniments of rapid chemical action, such as the evolution of beat, etc.) Antianaphylaxis can be produced to white-of-egg, as for serum, by the method of injecting small doses during the " incubation " time of anaphylaxis ; or by the injection of a minute dose, followed by a larger dose in 10 minutes, and so on for four doses. A guinea-pig, previously passively sensibilized, had o-qVo cx- injected intravenously ; in 10 min. another injection of ^^ c.c. ; in another 10 minutes, a third injection, this time of -fa c.c. ; and finally one of J c.c. Thereafter the injection of 2 c.c. of white-of- egg, non-diluted, caused uneasiness but nothing further. Antianaphylaxis can also be established by oral and by rectal feeding, in two to "four days. The state of anti- anaphylaxis is not so lasting as in the case of serum ; it lasts about three weeks, and two weeks where obtained by the oral or rectal method. Besredka and Bronfenbrenner also found that the reactions were strictly specific, and that the anaphylaxis produced by white-of-egg was in the main specific. Feeble reactions were given by the white-of-egg of pigeon and turtle-dove. The anti-anaphylactic is strictly specific. Similar experiments were made with heated white-of-egg, with like results. The one protects little against the other, so that they conclude that their chemical constitution is different. III. Serum-Globulin. — Turro and Gonzales have investigated the subject of anaphylaxis to determine the nature of the substance which causes anaphylaxis with blood serum. The globulins were precipitated from horse's serum, and the 218 PUBLIC HEALTH BACTERIOLOGY residual serum was kept. Guinea-pigs were injected with i c.c. of a 033 per cent solution of the globulins ; and 12 days after, it was found that the minimal test dose was 1 c.c. of a o-66 per cent solution (= 2 c.c. of the former solution). Anaphylactic shock developed rapidly, and there was a rapidly ascending paralysis, beginning in the hind limbs and causing death through asphyxia when the respiratory centres became affected, in 2 to 4 minutes. There were no convulsions, but in the male animals there was an abundant emission of serum. Another set of fresh guinea-pigs were given 1 c.c. of a 1 per cent dilution of the globulin-free serum ; and in twelve days thereafter, a test dose of the globulin solution produced no symptoms of anaphylaxis. (No mention is made of a test with the globulin-free serum itself.) An animal injected with normal serum, and later with the globulin solution test dose, showed intense muscular tremors, (the animal jumping about), but all ended in recovery. When the blood of a sensitized guinea-pig is mixed with globulin solution, and 1 c.c. injected into the jugular vein of a normal guinea-pig, the animal dies in 2 to 3 minutes with symptoms identical with those described in animals sensitized with pure globulins. They proceeded to study the anaphylactic poison, which they found is readily destroyed by oxidation and light, is dialysable, is thermostabile, and is soluble in alcohol and ether. They conclude that it is alkaloidal in nature, and should be considered a leucomaine, that is, a toxic substance produced in the living body by proteid metabolism, and not by bacterial action. IV. Classification. — The foregoing resume of the subject of anaphylaxis shows that it can be classified in the same manner as immunity into : — 1. Natural Anaphylaxis. 2. Acquired Anaphylaxis. And each of these into sub-groups, thus : — IMMUNITY AND ANAPHYLAXIS 219 Natural anaphylaxis depends on — a. Species of animal, e.g., cholera in man ; anthrax in cattle ; glanders in horses. b. Age, e.g., diphtheria in children ; erysipelas in elderly individuals. c. Individual, e.g., to white of egg, blood serum even by ingestion. (" One man's food is another man's poison.") Acquired anaphylaxis depends on — a. An attack of the disease, e.g., erysipelas and diphtheria. b. The injection of dead cells, e.g., tuberculin. c. The injection of nitrogenous matter, e.g., blood serum and white of egg. The subject of anaphylaxis has been dealt with here because of its intrinsic importance, and because it would seem to demand a restatement, in the near future, of the philosophy not only of infection but of medicine generally. The old idea of " diathesis " acquires anew its importance (having received direct experimental proof), and many apparently worn-out theories and old-fashioned explana- tions of disease may have new life put into them. CHAPTER XII. MICROCOCCI. These organisms consist of cells more or less globular in form and varying in size from 0-5 micron to 2 micra in diameter, but most measure about 1 micron (TTrVo mm., or ■2 5 thro °* an inch)- They are usually classified according to the mode of division or the resultant shape. Thus we have streptococci, staphylococci, diplococci, tetracocci (tetragenus), and sarcinae. None show endogenous spore formation, but of some it is alleged that they form arthro- spores. Most are non-motile, but a few motile species possessing flagella have been described (none pathogenic). STAPHYLOCOCCI. These were first demonstrated in pus by Pasteur in 1880 and Ogston in 1881, and in pure culture by Becker in 1883. Rosenbach in 1884 established specificity as cause of some forms of wound-suppuration and of osteomyelitis. They are so named from their growth in grape-like clusters. Several hundred species have been described, but the . chief varieties are : Staphylococcus pyogenes (aureus, albus, and citreus) ; Staphylococcus epidermidis albus ; Staphylococcus cereus (albus and flavus). The common characteristics are : Grape-like clusters of cocci, 0-9 micron in diameter ; non-motile ; non-sporing ; grow readily on most media ; stain readily ; Gram-positive ; gelatin-liquefying ; produce acid and clot in milk ; form indol ; reduce nitrates to nitrites ; show colour reduction with litmus, methylene-blue, and rosanilin (fuchsin) ; aerobes, but facultative anaerobes ; optimum temperature for growth, 280 to 300 C. Range from 8° to 420 C. Thermal death-point 30 min. at 8o° C. ; freezing useless. Cultures. — Put up (1) broth, (2) agar slope, (3) agar MICROCOCCI 221 plate, (4) gelatin stab plate and slope, (5) milk, (6) potato, (7) peptone water, (8) nitrate, and (9) sugar media. In broth, uniform turbidity with thin surface, pellicle ultimately settling as a heavy mucoid deposit, with a sour odour like weak butyric acid. On agar slope, abundant growth in 24 hours at 370 C. ; with smooth shining surface and resembling a streak of oil paint. Single colonies are circular. On agar plate, numerous small, shining, pin-head shaped colonies ; round, finely granular, with smooth edges, remaining discrete, and varying greatly in size. On gelatin plate, growth occurs readily at 200 C, and shows much the same as in agar. The colonies are not flat, but rise from the surface as the segment of a sphere. Liquefaction of the gelatin ; and gradually (after 48 hours or more), shallow saucer-shaped depressions are formed, which grow larger and finally become confluent. Lique- faction of gelatin by staphylococci is due to a ferment-like body elaborated by them and spoken of as " gelatinase " and which can be obtained apart from the cocci by filtration of cultures. It is an extremely thermolabile substance. In gelatin stab, a streak of growth is visible in 24 hours, and liquefaction begins at the top in 2 to 3 days, forming a funnel with flocculent deposit of the bacteria. Ultimately fluidification extends to the wall of the tube. In milk, coagulation takes place in 3 to 4 days with formation of lactic and butyric acids. On potato, growth is abundant, rather dry, and usually deeply pigmented. In peptone water, indol is formed. Pigment formation is best seen in serum or starchy media and aerobically only. It is insoluble in water but soluble in alcohol, chloroform, ether, and benzol. It is a C, H, and O compound, a " lipochrome ", or fatty pig- ment. Strong H2S04 changes it to green or green-blue. Toxic Products. — Endotoxins, hemolysins, leucocidins. Pathogenicity. — For man : abscesses, boils, carbuncles, endocarditis, osteomyelitis ; for animals : rabbits most, mice medium, guinea-pigs least. Immunization. — Take three-weeks-old culture, heat at 1 222 PUBLIC HEALTH BACTERIOLOGY 6o° C. for one hour ; inject ioo to 250 million of the dead staphylococci, repeating in 3 to 4 days later ; opsonins increased. Habitat. — Skin and mucous surfaces. Differentiation: (Gordon). — Staphylococcus pyogenes aureus clots milk, liquefies gelatin, at times produces green fluorescence in neutral red broth, reduces nitrates to nitrites, produces acid in Lemco media containing maltose, lactose, glycerin, and mannite. Staphylococcus epidermidis albus gives the same reactions, except that it does not produce acid with mannite. Houston's Lemco Medium : consists of distilled water containing 1 per cent of Lemco, 1 per cent of peptone, o-i per cent of sodium bicarbonate, and litmus solution to colour. Add 1 per cent of test substance (carbo- hydrate, etc.). STREPTOCOCCI. Ogston in 1881 first differentiated between the irregularly grouped staphylococci and the chain cocci or streptococci. Pure cultures were first obtained by Fehleisen in 1883 and Rosenbach in 1884. Named from tendency to form chains, and so the group includes micro-organisms which differ considerably from each other in cultural and pathogenic characters. The strep- tococci pathogenic to man mostly form chains of eight or more individual cocci, while the saprophytic varieties are apt to be united in shorter groups. On this basis streptococci have been divided into S. longi and S. breves, but the distinction is not a reliable one. Similarly, the streptococcus of ordinary pus formation was thought to be different from that of erysipelas. This is now proved not to be the case, and the two are regarded as one and the same. These statements enable one to attach the proper significance to the various names used ; to wit : S. pyogenes, S. erysipelatis, S. longus, S. brevis. S. conglomeratus is a variety, and is so called from its forming in broth culture minute granules composed of very long chains (identical with S. anginosus or scarlatinae) . Streptococci grow well on all the richer media, and better in broth made from meat or veal than from meat extract. Animal serum and MICROCOCCI 223 glucose render media more favourable for streptococci cultivation, and alkalinity 0-5 per cent. Streptococci are easily stained with the usual aniline dyes and are Gram-positive. They are non-sporing, non- motile, and non-flagellar. They (at least the pyogenic species) do not liquefy gelatin. Optimum temperature 37-5° C. Growth takes place at 150 to 200 C. (distinction from pneumococci). Aerobiosis is the suitable environ- ment for most races, but strict anaerobiosis does not prevent development in suitable media. Size 0-5 to 1 micron. Cultures. — In broth (bouillon) minute granules, which fall to the bottom and form a sparse powdery or sandy deposit. Diffuse clouding is rare. The long chain forms produce the coarser granules. In glucose broth, the rapid formation of lactic acid arrests development (1 per cent of sterile CaC03 obviates). In gelatin stab, in 48 hours a thin line forms which later is seen to be made up of minute rounded colonies of whitish colour reaching the size of a pin's head in 5 to 6 days. No growth on surface nor liquefaction. On agar plates, the colonies are small, greyish, and delicately opalescent, are round, and tend to remain separate in stroke cultures. In milk, ready growth with acid formation but no clot {in pyogenic varieties). On potato, no growth. On blood agar plates, most cause haemolysis and decolor- ization (difference from pneumococci). Ferment lactose, saccharose, and salicin ; not inulin in Hiss's medium. Thermal Death-point : 10 minutes at 540 C. Virulence varies ; greatest for white mice and rabbits. Habitat : skin and mucous membranes. Diseases : cellulitis, erysipelas, osteomyelitis, bronchitis, pneumonia, empyema, pericarditis, otitis, pharyngitis, tonsillitis, septic endocarditis, septicaemia, puerperal fever. Toxins: streptolysin (hemolytic). Immunization : variable results. Differentiation : (Andrews and Horder) . — Streptococcus pyogenes does not clot milk, nor give green fluorescence with neutral red broth, nor produce acid in Lemco media with rafhnose, inulin, coniferin, and mannite. It produces 224 PUBLIC HEALTH BACTERIOLOGY acid with lactose, saccharose, and at times with salicin ; it grows on gelatin at 20° C. ; it forms long chains, and is pathogenic to mice. Streptococcus salivarins clots milk, produces acid with saccharose and lactose, and at times with raffinose ; at times grows on gelatin at 20° C, and at times produces fluorescence. It is negative to the other tests, and grows in short chains. Streptococcus anginosus gives the same reactions as S. salivarius, except that it never produces acid with rafhnose ; it forms long chains, and it is pathogenic to mice. Streptococcus fczcalis (human) is positive to all the tests except production of acid with raffinose and inulin. It is non-pathogenic to mice, and it forms short chains. Streptococcus equinus (in horse dung) produces acid with saccharose, salicin, and coniferin, and grows on gelatin. It is otherwise negative ; forms short chains, and is non-pathogenic to mice. The pneumococcus forms short chains, is pathogenic to mice, clots milk, forms acid in lactose, saccharose, and raffinose, and at times in inulin. It does not grow on gelatin at 20° C. Examination of Pus. — By using solid media and method of 3 dilutions. (1) Gelatin plates : Three tubes are melted at 300 to 350 C. in a water-bath. Inoculate one with a loopful of pus, replug, and mix by rotating tube. This is tube of 1st dilution. Inoculate second tube with 3 loopfuls from 1st tube : tube of 2nd dilution. Inoculate third tube with 3 loopfuls from 2nd tube : tube of 3rd dilution. Now pour out all three tubes into dry sterile Petri dishes. Allow to set, and incubate at 200 C. (2) Agar plates : Same procedure, only melt at 1000 C. and cool to 400 C. before inoculating, and work quickly. Warm the plates. Incubate at 370 C. PNEUMOCOCCUS. The pneumococcus has been at various times called Streptococcus pneumoniae, Diplococcus pneumoniae, Micrococcus lanceolatus, and Fraenkel's pneumococcus. MICROCOCCI 225 The sputum of cases of acute lobar pneumonia, injected into mice or rabbits, produced more constantly a septi- caemia with fatal results than did the sputum of healthy individuals. In this " sputum septicaemia," lance-shaped cocci in pairs were most frequently found. Weichselbaum in 1886 examined 129 cases of all forms of pneumonia, and described four organisms which he found, the most frequently present being the one now known as the pneumo- coccus. It was present in all forms. The Streptococcus pneumoniae (now believed to have been a more vigorous pneumococcus) was next in frequency, then the pneumo- bacillus, and lastly the Staphylococcus pyogenes aureus. Description. — A small coccus, generally occurring in pairs, and surrounded by a definite capsule. The cocci are lance- shaped, like the flame of a candle; and in the pair have the pointed ends opposed. A close resemblance to a bacillus is thus formed. The capsule is characteristic in preparations from the sputum and tissues, but is only got in serum cultures, and is absent at times even in the sputum and in scrapings from the lung. The coccus is non-motile, non-flagellar, non-sporing. It is readily stained by the usual aqueous aniline dyes, and it is Gram- positive. The capsule is well shown in specimens stained by Gram and counter-stained : it takes the latter. Cultures. — Growth on the ordinary media is variable. From sputum it is best isolated by animal injection and cultures from the heart's blood. The best temperature is 37-5° C. Growth does not take place as a rule below 250 C. The colonies are like drops of dew. Blood serum and blood agar are the best media. In gelatin stab (when growth takes place), a row of minute dots appears, but no lique- faction. In broth, a slight turbidity results, which settles to the bottom of the tube as a dust-like deposit. Cultures rapidly die out, and virulence is quickly lost. In milk, growth is rapid, with acid and clot, and capsules are usually formed. Prolonged life is said to have been got in cultures on ascitic agar and blood-smeared agar. The pneumococcus ferments saccharose, lactose, ramnose, and inulin. Habitat. — The mucous membranes of the mouth, nose, throat, and conjunctiva. 15 226 PUBLIC HEALTH BACTERIOLOGY Products. — No soluble toxin yet isolated ; endotoxin by freezing and grinding.' Pathogenicity. — For mice and rabbits, very great ; for guinea-pigs, less ; for man, medium. Susceptibility is characterized by general septicemic infection ; resistance by occurrence of localized processes, e.g., in man : acute lobar pneumonia, pleurisy, empyema, pericarditis, menin- gitis, otitis media, etc. Isolation. — Inoculate a mouse at the root of the tail with a little of the suspected material. The animal dies in 24 hours, and its blood is swarming with typical, encapsuled lance-shaped diplococci, if pneumococci present. Immunity. — Agglutinins are formed, and agglutination is observed in 1-40 to 1-50 dilution with serum from pneu- monic patient, and most marked at time of crisis. Passive immunization with sera has been unreliable so far. A leucocyte extract with water has given encouraging results. Resistance. — Has been found alive in dried sputum after 55 days. Ten minutes at 520 C. is fatal. Very sensitive to weak solutions of disinfectants, but not in sputum. STREPTOCOCCUS MUCOSUS. This organism has been isolated from cases of menin- gitis, peritonitis, phlebitis, and parametritis, and from certain cases of pneumonia. It shows a marked tendency to form chains, but often appears in diplococcus forms. It is also capsulated, but never lance-shaped. It reacts to sugars as does the pneumococcus. It is patho- genic to white mice, but not so markedly to the rabbit as the pneumococcus. Growths are similar. It excites the formation of weak agglutinins, which can also in some cases agglutinate pneumococci. Also anti-pneumococcic serum frequently agglutinates it. These facts suggest a group relationship. MENINGOCOCCUS. First described by two Italian observers in 1884, but first cultivated by Weichselbaum from cases of cerebrospinal meningitis in 1887. It is a small coccus, very like the gonococcus, and like it occurring in pairs, MICROCOCCI 227 the adjacent sides being flattened like a coffee bean or two D's opposed to each other by the flat sides. In most cases it is present inside the protoplasm of the leucocytes in the exudation, the leucocytes being of the polymorphonuclear variety. Hence it has been called the Micrococcus intracellularis meningitidis. The variety of meningitis in which it is chiefly found is epidemic cerebrospinal meningitis, which is now a notifiable disease in a large number of districts. It is non-motile, non-sporing, non-flagellar, non-capsulated, and Gram-negative. It is found in the exudates, and specially in the spinal fluid obtained by lumbar puncture, and in such fluid it is well demonstrated in the cells by using Jenner's stain. It is easily stained by the usual dyes, and with methylene-blue stains irregularly. It is not readily cultivated on the ordinary media, and grows best on blood serum and ascitic agar. On blood serum, white "shining viscid colonies appear in 24 hours. Cultures readily die out. It ferments maltose and dextrose with acid production (distinction from M. catarrhalis) . Involu- tion forms occur. In the patient, agglutinins and opsonins are formed. Distinguish from M. catarrhalis, which is found in the nose, and grows at room temperature, whereas M. meningitidis is not easily grown below 250 C. The sugar test is also helpful. M. pharyngis also grows at room temperature and ferments maltose, dextrose, saccharose, and laevulose. M. mucosus gives slimy growths. Other Gram-negative cocci are chromogenic. GONOCOCCUS. First found in urethral pus and pus from ophthalmia neonatorum by Neisser in 1879. Cultivated by Bumm in 1885 on human blood serum. Diploforms very similar to the meningococcus, and like it may be found in pus, intra- but also extra-cellularly. The gonococci are non-motile, etc., and Gram-negative. They are mostly extra-cellular in chronic discharges, and may be rather scarce. They do not grow on gelatin or agar, but on serum or ascitic agar, and best at blood heat. Growth ceases below 300 C. Colonies appear within 48 hours, but may not until four days. They are readily killed at 420 C. They 228 PUBLIC HEALTH BACTERIOLOGY ferment dextrose but not maltose. A vaccine treatment lor gonococcus has been used with success in chronic cases. An emulsion of gonococci in sterile salt solution (0-85 per cent) is heated to 650 C. for 1 hour. Inject 300 million, and give dose every 7 to 10 days, increasing to 1000 to 1200 million. MICROCOCCUS TETRAGENUS. Found in 1881 by Gaffky, in pulmonary cavities. Usually non-pathogenic in man. Grows well on ordinary media, clouds broth evenly, does not liquefy gelatin, clots milk with acid formation, is Gram-positive, and is cap- sulated in the body. It is pathogenic to white mice, causing septicaemia ; slightly so to guinea-pigs and rabbits, and non-pathogenic to house mice and rats. It has been described as the cause of abscesses, on one occasion of meningitis, and on another of septicaemia. MICROCOCCUS CATARRHALIS. Found in patients suffering from catarrh of the upper respiratory tract. Its chief claim to attention is its similarity in staining and morphology to the meningo- coccus and gonococcus. From the latter it is distinguished by its rapid growth on the ordinary culture media. From the meningococcus, with which it may be found in the nasal passages, it is similarly distinguished, but here the difference is one of degree only. The sugar tests are very helpful. Dextrose Maltose Laevulose Sacchar- ose Lactose Galactose Meningococcus . . Gonococcus M. catarrhalis . . M. pharyngis sice. + + O + + O O + O O O + O O O + . O O O O O O O O M ICROC DCCUS MELI TENSI! 3. This organism was first described by Bruce in 1887, as present in the spleen of patients dead of Malta (or undulant, rock, Mediterranean, or Neapolitan) fever. MICROCOCCI 229 This is an endemic pyrexial disease, occasionally pre- vailing as an epidemic, having a long and indefinite duration and an irregular course, with, almost invariably, pyrexial relapses of an undulatory type. The illness may last from 20 to 300 or more days, averaging 60 to 70 days, and having a mortality of about 2 per cent. The chief mode of spread formerly was the ingestion of goat's milk, in which the micrococcus was being excreted. The urine of patients is also infectious. The Mediterranean Fever Commission conclude that Malta fever is a septi- caemia, in which the specific organism can be recovered from the peripheral blood, the urine, and the faeces. Infection is not conveyed by the sputum, sweat, breath, or skin-scrapings of patients. It does not take place if contact is limited to skin surfaces only, and if urinary and faecal contamination are excluded. It is probably occasion- ally conveyed by sexual intercourse. About 10 per cent of the Maltese goats excrete the micrococcus, and 50 per cent give a positive agglutination reaction. Goat's milk is prob- ably the prime source of the disease in most cases if not in all. Characteristics. — Micrococcus melitensis is a very small coccus, 0-3 micron in diameter, occurs singly or in pairs, and at times in short chains. It is non-motile and Gram- negative ; does not ferment glucose (unlike ordinary strepto- coccus), and renders milk slowly alkaline. It is easily stained. Some observers describe it as a minute bacillus. Cultures. — On agar, it forms minute transparent colonies likened to dew-drops, but only after 2 to 3 days' culture at 37-5° C. On gelatin, liquefaction is not produced. In broth, no indol is formed, nor odour ; but a slight turbidity. On potato, a moist transparent growth is formed. Agglutination of the organism by the patient's serum is shown after the fifth day. In some cases it persists for years. Resistance. — Is a vigorous organism and resists desicca- tion for weeks. Malta Fever. — Other symptoms of the disease are : shifting rheumatic-like pains, profuse sweatings, con- stipation, local neuritis, emaciation, and almost always enlarged spleen. The micrococcus may be isolated by splenic puncture or from the blood. CHAPTER XIII. NON-SPORING BACILLI. THE COLON-TYPHOID-DYSENTERY GROUP. This is 'a large' group, which includes the colon bacillus and its allies ; the typhoid bacillus ; paratyphoid bacilli ; dysentery bacilli and allies ; and Bacillus faecalis alcaligenes. Closely related to the group (but not properly within it) are the B. lactis aerogenes, B. mucosus capsulatus (Fried- laender's bacillus), and B. proteus. \ All the members of the group are bacilli and are very similar morphologically, but exhibit minor differences insufficient to permit of accurate diagnosis from mor- phology alone. All are non-sporing, non-liquefying of gelatin, Gram-negative, and grow well at room and body temperatures on artificial media. They are distinguished from one another by a careful cultural and biological study, to wit : reactions in special media (e.g. the fl-ag-in-ac group of reactions : — fluor- escence with neutral red, acid and gas with lactose, indo] with peptone water, acid and clot in litmus milk) ; motility and flagella ; and reactions with specific immune sera (chiefly agglutination) . Bacillus Coli Communis is a name which stands for a group of organisms, one member of which was first described by Buchner in 1885. The one taken as a type of the group was obtained from the stools of a breast-fed infant, and was described by Escherich in 1886 as the Bacterium coli commune. It is now usually designated B. coli (Escherich). It is widely distributed in nature and has been isolated from air, water, and soil, but is found most abundantly and constantly in the intestinal tract of man and of many of the higher animals, from which habitat it probably rinds its way into soil, water, and air. Its chief characteristics are : short plump rod, 2 to 4 micra long and 0-4 to 07 micron broad (very short oval and coccus-like forms are found, NON-SPORING BACILLI 231 especially in the animal tissues) ; grows well on usual media ; curdles . milk with acid production in forty-eight hours ; forms acid and gas with dextrose and lactose (gas = hydrogen and carbonic acid gas in proportion of 2 to 1) ; some ferment saccharose ; some are weak and aberrant ; not usually pathogenic (agonal or post-mortem invasion excluded) ; sometimes causes peritonitis, cholecystitis, pyelitis, and cystitis ; can precipitate cholesterin from solution (hence may cause gall-stones) ; cannot peptonize native proteids (casein and egg albumen, etc.). Cultures. — In broth : uniform turbidity. In gelatin stab : growth along whole line of stab and film-like abundant growth on surface, but no liquefaction. On gelatin plate : surface colonies are apt to show the typical grape-leaf formation. (In gelatin stab, a few gas bubbles may form, see later.) On agar slope : a dense glistening white? or greyish growth. Same on blood serum. On agar plate : surface colonies show grape-leaf structure. On potato : abundant growth, at first greyish- white, turning later to yellowish-brown. Cultures are characterized by a peculiar foetid odour, not unlike that of diluted faeces. Grows well on media containing urine and bile. In peptone water : forms indol. In milk : acidity and clot. In lactose litmus agar : the medium becomes red along stab, and gas bubbles appear. In carbohydrate media : acid and gas are formed in presence of glucose, lactose, laevulose, galactose, maltose, raffmose, mannite, dulcite, and sorbite ; and occasionally in saccharose (cane sugar) and in the gluco- sides, salicin and arbutin. Some varieties change neutral- red, first to a rosy-red and then to a green fluorescence (in glucose, broth) ; and most reduce nitrates to nitrites. Aerobe, but facultative anaerobe. Motile, having from 4-12 peritrichal flagella. B. Typhosus was discovered by Eberth in 1880 in the spleen and mesenteric glands of persons dying of typhoid or enteric fever. In such sections the bacilli occur in groups, scattered individuals being rare. Gaffky in 1884 first grew it in pure culture and studied its characters. Characteristics. — Short plump rod with rounded ends7 1 to 3 micra long and 05 to 08 broad ; actively motile ; numerous peritrichal flagella (10 to 14) ; growth less 232 PUBLIC HEALTH BACTERIOLOGY luxuriant than B. coli ; Gram-negative ; produces acid but no gas in dextrose broth and agar, no change with lactose or saccharose, no clot in milk, but in litmus milk slight acid at first, from small quantity of monosaccharid present ; later, deep blue from formation of alkali ; no indol in peptone water ; on • potato slight moist glistening growth, becoming dull and velvety. Cultures. — In broth : uniform turbidity. In gelatin stab : growth to bottom of stab, and on surface as a thin leaf- like film or pellicle with irregular wavy margin. In gelatin plate, the colonies are smaller, more delicate, and more transparent than those of the colon bacillus of the same age. In agar plates, in twenty-four hours, small greyish colonies are formed, at first transparent but later opaque. On acid potato : slight moist glistening growth. Forms acid but no gas with dextrose, laevulose, galactose, maltose, mannite, and dextrin ; but no change with lactose and saccharose. In Hiss's tube medium (agar, gelatin, dextrose, Liebig's extract, salt, water), it gives uniform clouding owing to its motility, but no gas ; whereas dysentery bacillus grows only along stab, and colon bacilli form sky-rocket-like figures, and the medium is broken with gas bubbles. Optimum temperature, 370 C. ; range 15 ° to 41 ° C. Resistance like most non-sporing organisms, 30 min. at 6o° C., or 2 to 3 min. at ioo° C. In ordinary tap or distilled water it is usually found dead in three weeks (Frankland). The researches of Dr. A. C. Houston have shown that in the crude river water derived from the Thames, raw typhoid bacilli die out in one to two weeks, whereas cultivated typhoid bacilli may persist for five weeks. On this basis, the storage of water for thirty days, preliminary to filtration, is looked upon as of prime importance in all waters derived from sources polluted by sewage. Does not multiply in water, even when impure. Is by preference a parasite, and when found outside the body can be usually traced to sewage of typhoid patient or convalescent (carrier). In natural bodies of water it retains its vitality for at least four to five days, and in sterile water it may be found surviving for three months. History of typhoid epidemics from sewage-polluted water NON-SPORING BACILLI 233 shows that danger is chiefly to be feared when pollution is recent. In soil and faecal matter, however, duration of vitality is more prolonged (five months in privy refuse, and fourteen days after being spread on ground), but no genuine multiplication proved. This suggests one mode of pollution of surface water and surface wells after rains, by the washing of dormant bacilli into the same, and to the danger of using human excrement for manuring vegetable gardens and fields near water sources (Lincoln, 1905). Air-borne infection is rare. Sewer air is not regarded as an actual cause so much as a predisposing one. Pathogenicity. — For man : enteric fever, typhoid fever, abdominal typhus (German), la fievre typhoide (French). For animals : do not multiply, and same effects by injection of dead bacilli, as that due to endotoxins. Chimpanzees have been infected by food, and have shown characteristic lesions. Intraperitoneal injections produce a short acute illness with pyrexia, etc., but non- specific. Specificity has not been absolutely proved according to Koch's postulates. In enteric fever the bacilli are present in the blood, the bowel, the urine, the sputum and the rose spots. Toxins are intracellular. Immunity usually follows one attack, and is due to bactericidal and bacteriolytic bodies. Active Immunization is accomplished in animals by a first injection of 1 c.c. of a broth culture heated for ten minutes at 6o° C, followed in five or six days by a larger dose, and so on, until finally living cultures are injected in considerable doses without serious consequences. Wright's vaccination against typhoid has been used extensively in the British army. He uses a strain of bacillus standardized by passage through guinea-pigs, and sterilizes the culture by heating to 6o° C. for five minutes. The first injection is of an amount of bacilli fatal to 100 grm. of guinea-pig, or alternatively, 750 to 1000 million of dead bacilli. The second injection given eleven days later should be double the first. The first dose is followed by tenderness and swelling locally and at the adjacent lymphatic glands, and some pyrexia, all of which usually subside in twenty-four to forty-eight hours. The method 234 PUBLIC HEALTH BACTERIOLOGY is believed to reduce the case incidence and the mortality among vaccinated persons attacked. Vaccine treatment of typhoid fever on similar lines has been tried by Leishman and Smallman, using' one-fifth of the dose used for protective inoculation. If the tempera- ture fall, the injection may be repeated every four days. Antityphoid serum has been used, but results are equivocal. Agglutination. — The blood or blood serum of an animal previously inoculated with typhoid bacilli, when added to a suspension of living and motile typhoid bacilli, causes the latter to become motionless and aggregated (clumped). This reaction is one given in many diseases after the attack — a reaction of immunity ; but in enteric fever it is given during the attack — a reaction of infection. The reaction is specific in high dilutions, but not absolutely so. Group bacilli are clumped, but in lower dilutions : for example, serum clumping typhoid bacilli in a dilution 1-40,000 will clump paratyphoid bacilli in a dilution 1-1000, and coli bacilli in 1-500. The reaction is used for : (1) Diagnosis of disease (Widal's reaction) ; and (2) Diagnosis of bacteria (Grtieber's reaction). 1. Widal's Reaction. a. Microscopic method. — Requirements : An eighteen to twenty-four hours' culture in broth of undoubted B. typhosus. Blood or serum. Platinum loop, a hollow ground slide, an ordinary slide or a watch-glass, cover- slips, a one-sixth-inch lens, and vaseline. Procedure. — 1. Test motility of bacilli and absence of clumping by putting up a hanging drop of culture. If clumps, filter. If movements sluggish, warm. 2. Take a clean dry slide and put on it nine loopfuls of sterile broth arranged in a small circle. Put one loopful of serum in centre of circle and mix. Dilution 1-10. Now put one loopful of 1-10 dilution on a clean slide or hollow slide, mix with three loopfuls of sterile broth, and finally with one loopful of culture. Dilution is now 1-50. 3. Mount one loopful on a cover-slip, invert over hollow slide, and examine. Examine again in fifteen minutes and after one hour. If reaction is positive, the bacilli will be arranged in groups and be non-motile, and between the clumps will be clear spaces. NON-SPORING BACILLI 235 Or the serum may be diluted by means of a graduated pipette, such as a leucocytometer pipette, or by using a capillary pipette which is rilled to a certain mark and emptied into a watch-glass, and the desired number of the full of the pipette of bouillon added and mixed. The mixture of a pipette-full of the diluted serum and of the bacillary emulsion is then examined, the final dilution being double that of the diluted serum. b. Macroscopic method or Sedimentation. — Take a range of test tubes, 5 cm. x 0-5 cm., and put into each 1 ex. of various serum dilutions and 1 c.c. of bacillary emulsion. Also put up a control with 1 in 20 normal serum. Plug, and keep vertical, and either incubate for three hours at 370 C, or keep at room temperature for twelve to twenty- four hours ; or first incubate and then keep for twenty-four hours, reading the results at both stages. The result can be controlled by microscopic examination of the super- natant fluid. The statement of the result should comprise all the conditions of the experiment, namely : kind of test (hanging- drop or tube), dilution of serum, times of observation, and intensity of reaction (complete, medium, or nil agglutin- ation). The test is also given by dead bacilli. Young cultures are used to prevent spontaneous agglutination. It is not always convenient to have young cultures, and so the following suspension of dead bacilli (which keeps well but agglutinates tardily though easily), may be used : To a twenty-hours' broth culture, add 1 per cent formalin, incubate for two days at 370 C, pour off the fluid from the precipitate and store in an ice chest. Interpretation of Results. — A positive Widal reaction may be due to : (1) An attack of typhoid fever (especially if increasing) ; (2) A previous attack ; (3) An attack of paratyphoid fever ; (4) Some other disease (jaundice and tuberculosis) . A negative Widal may be due to : (1) Too great or too little infection ; (2) Disease not typhoid ; (3) Test applied too early (before eighth day) ; (4) Inhibition phe- nomena (some sera agglutinate in a dilution 1-100 and not in 1-20). 236 PUBLIC HEALTH BACTERIOLOGY 2. Grueber's Reaction. — Consists in determining the race of a bacterium by testing it against the blood serum of an animal immunized to an organism of known race. The reaction is the converse of the Widal one, but high dilutions (i-iooo) must be used to avoid error due to group agglutinins. It is open to the fallacies that some bacilli are inagglutinable, though belonging undoubtedly to an agglutinable race (some typhoid bacilli), and bac- teria giving a negative result may nevertheless have very similar pathogenic characters to those giving a positive result. Principal Channels of Infection. — Water, milk, ice-cream foods, oysters, mussels, water-cress, lettuces, radishes, flies, dust, contact, chronic germ-carriers. Paratyphoid Bacilli (also called paracolon bacilli) — are the probable cause of a disease clinically resembling mild typhoid (about 3 per cent of cases treated as typhoid are probably paratyphoid). Onset is usually sudden, with chills; mortality is low (2 per cent), and on post-mortem examination no characteristic ulceration of Peyer's patches is observed. Two chief varieties are described, namely, paratyphoid " A " and paratyphoid " B." Of these B is the more widely distributed in nature, and is more pathogenic to animals, and is believed by some to be the same as B. enteritidis (Aertryck) and B. typhi murium (mouse typhoid) . In cultures A resembles the typhosus ; and B the colon bacillus ; and fermentative reactions like- wise, except in milk, where A gives slight permanent acidity, and B slight acidity which after the third day gives place to alkalinity. Cases of illness due to A, resemble mild typhoid ; and to B, are allied to food-poisoning with severe gastro-intestinal symptoms. Organisms of this group form endotoxins which are heat resisting, and there- fore the ingestion of cooked food containing the bacilli may produce severe disturbance, thus relating them to Gaertner's bacillus (B. enteritidis). None form indol. All give fluorescence with neutral-red. These bacilli are motile, flagellar, show agglutination phenomena, and ferment dextrose, dulcite and mannite, but not lactose nor saccharose. They are present at times with the B. typhosus, and may thus produce a mixed infection. NON-SPORING BACILLI 237 B. Enteritidis (Gaertner).— In 1888 the flesh of a diseased cow was sold for food in a Saxony village. Gastro- enteritis followed the ingestion of the meat in the case of fifty-seven people. One young man ate 800 grm. (nearly 2 lb.) of the raw meat, and died in thirty-five hours. From his spleen and blood Gaertner isolated an actively motile bacillus, closely resembling the typhoid germ ; and he obtained the same organism from the flesh of the cow. Similar bacilli have since been found in other outbreaks of meat-poisoning. Gaertner's bacillus is very pathogenic to laboratory animals, causing an intense hemorrhagic enteritis. The symptoms are due to endo- toxins which are heat resisting, so that boiling does not readily destroy the toxicity. It grows more rapidly on gelatin than B. typhosus ; forms no indol, but ferments dextrose, with formation of acid and gas. Closely related to the bacillus of Gaertner are the hog cholera bacillus and the B. psittacosis. The latter was first isolated in Paris in 1892 in a highly fatal pneumonia-like illness (49 cases, 16 deaths), which was traced to sick parrots from South America. B. icteroides and B. typhi murium are also of this group. Danysz's virus is supposed to consist of B. enteritidis (iErtryck and Gaertner) . B. Dysenteriae, or Shiga's bacillus. First isolated from the stools of patients (in Japan) suffering from acute dysentery, in which no amoeba could be found. The bacillus was found by examining the stools for an organism which would agglutinate with the serum of the patients. In 36 cases one and the same organism was found to meet the test, and it was not found in the dejections of healthy persons or persons suffering from other diseases, nor did it agglutinate with their blood serum. It is now recognized as the specific cause of acute epidemic dysentery of temperate climates. Since then, several bacilli have been isolated in different parts of the world, all related to Shiga's bacillus but giving a variety of reactions to carbohydrates and to immune serum. Kruse isolated his organism from " pseudo-dysentery of the insane," and Flexner from dysen- tery in the Philippines. They all ferment dextrose but without gas formation. In milk, first slight acidity and 238 PUBLIC HEALTH BACTERIOLOGY then increased alkalinity is produced. The Shiga-Kruse group produce no indol, but the Flexner group do. Short rod, non-motile, non-liquefying, non-Gram-staining, aerobe and facultative anaerobe. Bacillary dysentery is an ulcerative colitis with little ten- dency to form liver abscess. It is spread like enteric fever, and is a scourge of armies as it formerly was of asylums. Animals are easily killed by injection, but show no charac- teristic lesions in the intestine, though the latter have been obtained by feeding experiments. The Shiga-Kruse bacilli form a soluble toxin which is very fatal to rabbits, and is resistant up to 700 C. This toxin causes profuse diarrhoea, and later paralysis, when injected intravenously into rabbits, being apparently excreted by the colon and caecum. Similar bacilli have been isolated in the summer diarrhoea of infants. An anti-toxic serum has been prepared from horses. B. Faecalis Alcaligenes resembles B. typhosus morphologically and culturally, but is non-pathogenic. Cultures. — Gives a luxuriant growth on potato ; forms no acid with glucose, but alkali with milk whey and mannite. It is an occasional inhabitant of the ileum and colon. Other organisms which have been described in connection with the colon-typhoid-dysentery group are : B. nea- politanus (in a choleraic disease) ; B. acidi lactici, of Hueppe ; B. lactis aerogenes (resembles Friedlaender's diplobacillus), and other bacilli present in milk, which appear in the faeces of milk-fed persons. Voges-Proskauer Reaction is not given by B. coli nor by any of the above, but by B. lactis aerogenes, B. cloacae, and B. oxytocus perniciosus. Inoculate glucose-peptone solution and grow for 3 days ; add KOH ; stand for twenty-four hours. Red colour. Differentiation of B. Typhosus from B. Coll The problem is to find a medium which will favour the development of B. typhosus and B. coli and yet will differentiate them. The usual method is to use coloured media + inhibitory agents or favouring agents. NON-SPORING BACILLI 239 1. Drigalski and Conradi's Medium. — This is a meat broth (1-5 lb. per litre) to which, besides the peptone and salt, i per cent of mitrose and 3 per cent of agar are added and dissolved. Thereafter litmus solution is used to dissolve pure lactose (quantity used = half quantity of agar) , and the whole is added to the hot agar fluid. Render alkaline with sodium carbonate solution, and add a solution of crystal- violet. The medium must not be overheated or the lactose may be changed. It is a solid medium and is, shortly, a lactose-nutrose-agar. The crystal-violet restrains the saprophytes. In twenty-four hours B. coli colonies are red ; 2 to 6 mm. in diameter, and non-transparent. B. typhosus colonies are blue ; 2 mm. in diameter, and glassy and dew-like. The plates are inoculated by smearing the surface with a glass rod, dipped in, say, diluted faeces. 2. Endo's Medium. — This is a 3 per cent agar neutralized and then alkalinized with NaOH, and lactose and fuchsin (basic) solution added, and then Na2S03 solution until decolorized. Put into test tubes (15 c.c), sterilize, and keep in dark. When using, pour plates, and inoculate by surface smears, when in twenty- four hours B. coli colonies are red, and B. typhosus colonies are colourless. 3. Loeffler's Medium. — This is a 3 per cent agar to which malachite-green is added, and this retards growth of B. coli. B. typhosus colonies are minute glistening points ; later, they colour agar yellow. 4. Hoffman and Ficker's Medium. — Convert water sample into medium by adding : caffein 2-5 per cent (restrains B. coli) ; nutrose 1 per cent ; and crystal-violet o-ooi per cent (restrains saprophytes). Incubate at 370 C. for not more than 12 hours. The B. typhosus can then be isolated on plate media. 5. MacConkeys Media — Have been already described on page 153. The bile-salt assists growth of B. coli and B. typhosus, and hinders others. Where neutral-red is used, acid formation changes it to a rose-red. 6. Bile Medium (for blood). 7. Hiss's A gar-Gelatin Media, see p. 232. 240 PUBLIC HEALTH BACTERIOLOGY Table of Characteristics. B. Coli B. Typhosus Paratyphosus A B Dysenteriae jFajcalis A B I Ale. Motility + + + + — — + Gram negative negative negative negative neg. Peptone water indol — — — — indol — Glucose A + G A A+GA+G A A nil Lactose A + G — Saccharose — — — — _ _ j _ Milk (litmus) A + Clot ( A (alk. in 4 A (alk. in A (alk. in | Alk. ! to 10 days) 3 days) 3 days) Gelatin non- non- non- non- 1 non- liquefying liquefying liquefying liquefying llique- Pathogenic : For man slight distinct distinct distinct nil ,, animals do. less more more j nil Short Table of Carbohydrate Reactions. 1 Glucose Lactose Saccharose Dulcite Mannite B. typhosus . . A A B. coli . . + + — + + B. acidi lactici ! + + — — + B. paratyphosus + — — + + A signifies acid ; + acid and gas ; - nil. CAPSULATED BACILLI. In this group are classed bacilli which are non-motile, and capsular ed in certain cultures, but otherwise resemble the Bacillus coli. Bacillus Pneumoniae. — Otherwise called pneumo- bacillus, Friedlaender's bacillus. Was first described by Friedlaender in 1882 as the cause of acute lobar pneumonia. It was first called a micrococcus, and was confused with Fraenkel's pneumococcus, but was later recognized as a short bacillus. It is the cause of pneumonia in about 7 per cent of the cases. It is taken as the type of a group, " the mucosus capsulatus " group, and is also called B. mucosus capsulatus. NON-SPORING BACILLI 241 Characteristics. — A short plump bacillus with rounded ends, but showing some very long forms (o-6 to 5 micra long by 0-5 to i;5 micron). The short thick forms are mostly found in animal tissues, and at times are almost coccoid. In sputum it shows a capsule, and in other preparations from the body. It occurs in pairs, and hence has been called a diplobacillus. It may also form short chains. It is non-motile, non-flagellar, non-sporing, non-gelatin- liquefying, and non-Gram (i.e., Gram-negative). Cultures. — It grows readily on ordinary media and in gelatin at room temperature. It grows well on acid or alkaline media, is aerobic, and facultatively anaerobic. In broth : rapid abundant growth with a pellicle, general clouding, and later a stringy sediment. On agar : sticky mucus-like colonies of a greyish-white colour. In gelatin stab : a white line of growth at first, but with increasing growth at surface a ' nail-head " appearance is produced. This was at one time thought to be peculiar to Friedlaender's bacillus. On potato : abundant, somewhat brownish growth. In peptone water : no indol formation. In milk : abundant growth with capsule formation. Acid and clot are slowly formed. Pathogenicity. — For man : pneumonia of a severe and fatal type ; ulcerative stomatitis and nasal catarrh ; acute tonsillitis ; in antral suppurations and in foetid coryza ; and, on rare occasions, in septicaemia. For animals : a mouse injected at the root of the tail dies in two days of septicaemia. It is also pathogenic for guinea-pigs ; less so for rabbits. Distinction from THE PNEUMOCOCCUS. Pneumococcus Pneumobacillus Growths on ordinary media on gelatin , , in milk Staining Sparse Almost none . . Acid + clot Gram-positive . . Good Nail-head growth Acid + clot (late) Gram-negative Allied bacilli are : B. ozaenae, found in foetid nasal catarrh, which is scarcely separable from B. mucosus, 16 242 PUBLIC HEALTH BACTERIOLOGY capsulatus ; bacillus of rhinoscleroma. Both these bacilli differ only in not fermenting dextrose. B. Lactis Aerogenes — Is a widely distributed organism, and was isolated by Escherich in 1885 from the faeces of infants. It is almost constantly present in milk, faeces, sewage, and water. It differs from B. coli, which is found in like circumstances, in being non-motile and non- flagellar, in possessing a capsule in milk cultures, in fermenting saccharose and starch but not dulcite ; and in not forming indol. Cultures. — It grows readily on all media. In broth, it forms a pellicle, and causes general clouding. On agar and gelatin, it forms a heavy white growth. In gelatin stab, it gives a nail-head growth. On potato, it grows well and forms gas from the starch. In Milk, acid formation and coagulation are rapid. The clot is not digested by the bacillus, and in ordinary souring of milk the germs present which produce proteolytic ferments have their growth restrained by the large amount of lactic acid formed by the more rapid action of B. lactis aerogenes. It is scarcely pathogenic, though flatulence in infants has been attributed to its action, and a cystitis in which gas was formed in the bladder, associated with an acid urine. For animals, its patho- genicity is not property established, the reports being contradictory. It is an aerobe, but a facultative anaerobe. Optimum temperature, 250 to 300 C. B. lactis aerogenes is distinguished from Friedlaender's bacillus by its invari- able and rapid curdling of milk; but some authorities consider it to be identical with that organism. BACILLUS ACIDI LACTICI (HUEPPE), This bacillus is present in milk, which it curdles and acidifies. It does not ferment saccharose or dulcite. B. acidi lactici (Leichmann) is believed to be really a streptococcus, the Streptococcus lacticus (Kruse), which Heinemann states is a variety of the Streptococcus pyo- genes. It is present on the cow's hide, in cow dung, and in milk from the first stage of milking. NON-SPORING BACILLI 243 MINUTE BACILLI. Under this heading may be conveniently grouped the bacilli, of influenza, of acute epidemic conjunctivitis, and of whooping-cough. All are Gram-negative. Bacillus Influenzae — Was first described in 1892 by several observers, and has been called after one of them the Pfeiffer bacillus. It is very small even among micro- organisms, being only from 0-5 to 1-2 micron long by 0-2 to 0-4 micron thick. A tubercle bacillus, 3 micra by 0-3 micron is thus equal in length to several (3 to 6) influenza bacilli placed end to end. It is then a small bacillus, of irregular length, having rounded ends, rarely forming chains, non-motile, non- sporing, Gram-negative, and not growing on gelatin or at room temperatures. It is not easily stained with the usual dyes ; best with 10 per cent aqueous fuchsin, or Loerfler's methylene-blue, 5 minutes of either. The bacilli form irregular clusters ; occasionally polar staining is noticed. Cultures. — It is not easily cultivated, growing only in the presence of haemoglobin. This is obtained on the ordinary media by smearing them with some blood drawn from the finger, or by mixing melted agar with fresh blood. The blood of the pigeon may be used. The medium is inoculated with the sputum coughed up from the bronchi, avoiding mucus from the mouth. Colonies appear in 18 to 24 hours, as minute transparent drops, colourless, and likened to drops of dew. Growth ceases in 2 to 3 days. Frequent subculturing and storage at room temperature are needed to keep the cultures alive. It is aerobic, and shows no growth under strict anaero- biosis. It is readily killed at 6o° C. ; and by drying, in a few hours. Dies in culture media within a week. Its usual habitat during an epidemic is the nasal passages and bronchial tubes. It is said to remain in these places after recovery from the attack, and to persist for years. The immunity produced by an attack of influenza is very* short. A pseudo-bacillus, differing only in its slightly larger size and its growth in threads, and showing involution forms, has been described, but its differentiation is doubtful. 244 PUBLIC HEALTH BACTERIOLOGY Koch-Weeks Bacillus. — A bacillus similar to the above, but longer and more slender, was described by- Koch in 1883, and by Weeks in 1887, in connection with an epidemic form of acute conjunctivitis. Cultures. — It grows best on serum agar, and at 370 C ; the colonies appear in 36 hours as dew drops. The disease is characterized by a muco-purulent discharge, hyperaemia of the whole of the conjunctiva, and swelling of the lymph follicles of the lids, which show through the palpebral conjunctiva as slightly raised pinkish-grey bodies, a half to one millimetre in diameter. A film made from the discharge and stained with Loeffler's methylene- blue, shows the bacilli. The affection is very contagious, and to prevent epidemics in schools, common face-towels should be rigorously prohibited. Bordet-Gengou Bacillus. — These observers found a small ovoid bacillus in the sputum of a child suffering from whooping-cough. Culture. — In 1906, six years later, they succeeded in cultivating it on a special medium, after failing with ascitic agar and blood agar. This medium is a glycerin extract of potato with 4 per cent salt and 2-5 per cent agar, to which is added an equal quantity of defibrinated human or rabbit's blood. On this, inoculated from sputum, the colonies appear within 48 hours, and are small, greyish, and rather thick. In subcultures, they give a more luxuriant growth, and can then be grown on blood agar and in ascitic broth, in which it causes a viscid sediment but no pellicle. It is strictly aerobic, and grows moderately below blood-heat. It remains alive in culture for as long as two months. Specific agglutinins are developed in immunized animals, which serve to distinguish it from B. influenzae. The washed and dried bacilli ground in a mortar and injected into a rabbit intravenously, usually kill it in 24 hours. Specific complement fixation has been used by Bordet and Gengou to prove the identity of the bacillus, using the serum of an infant suffering from whooping-cough. The organism is present in the sputum in the early stages in predominating numbers, but later it is swamped by others. It is scattered among the pus cells, NON-SPORING BACILLI 245 and at times is intracellular. It is extremely small and ovoid, so that it is readily mistaken for a micrococcus. It is slightly larger than the bacillus of influenza, and more ovoid. It is Gram-negative, stains with Loeffler's methylene-blue, dilute carbol-fuchsin, or aqueous fuchsin. Toluidin blue in a special medium is advised. (Toluidin blue 5 grm., alcohol ioo c.c, water 500 c.c. ; dissolve ; add of 5 per cent aqueous carbolic acid solution, 50 c.c. ; stand two days ; filter.) Bacillus of Ducrev — Is a small bacillus (1 to 2 micra X 0-5 micron), regularly found in soft chancre or chancroid. It is non-motile, non-flagellar, non-sporing, Gram-negative, and is found at times inside the leucocytes. Culture. — It only grows on whole blood agar, and dies off at room temperatures. Colonies show in about 48 hours. Inoculation of pure cultures on the skin produces typical chancres in 4 to 6 days. Zur Nedden's Bacillus. — A small slightly curved bacillus (1 micron long) which has been isolated in ulcera- tive conditions of the cornea. It grows well on the ordinary media. It is non-motile, Gram-negative, and does not liquefy gelatin. It clots milk, forms acid with glucose, but no indol in peptone water. Culture. — It grows well on potato. MORAX-AXENFELD DIPLO-BACILLUS. This is a short, thick bacillus with rounded ends, found in a chronic eye condition called " angular conjunctivitis," in which there is slight redness of the edges of the lids, especially at the angles. The ocular conjunctiva is seldom affected. It mainly affects adults, and chiefly women. There is rarely any corneal trouble. It was described by Morax in 1896, and by Axenfeld a year later. The diag- nosis is easily made, by taking a smear of the small bead of pus which gathers through the night at the angles, and staining with the usual dyes. The bacillus is easily stained, and is Gram-negative. In stained films, short and long chains of polar stained bacilli are seen. Culture. — It grows best on Loeffler's blood serum at 370 C, causing small pits of liquefaction in 48 hours. 246 PUBLIC HEALTH BACTERIOLOGY DIPHTHERIA BACILLUS. Klebs first described the bacillus in the throat mem- brane in 1883, and Loeffler first cultivated it in 1884 ; hence called the Klebs-Loeffler bacillus. The toxins pro- duced by it were investigated by Roux and Yersin in 1888- 89, and the antitoxins by Behring and Kitasato in 1890. Description. — A slender bacillus, 1 to 6 micra long and 0-3 to i-i micron broad. From the throat they are mostly 4 to 5 micra long. They are rarely of uniform thickness throughout, showing club-shaped thickening at one or both ends. They are straight or slightly curved, stain deeply with methylene-blue, often showing granules more darkly stained, so that a dotted, beaded, or striped or barred appearance results. The longer individuals often have a strong resemblance to short chains of streptococci. In 18-hour-old cultures, many of the bacilli show on staining deeply stained oval bodies situated most frequently at the ends, the so-called " polar " or " Babes-Ernst " bodies. These were first regarded as spores, but are now considered to be chromatic granules. B. diphtheriae is an aerobe. Is non-motile, non-sporing, non-flagellar, and non-liquefying of gelatin. It is Gram-positive. Cultures. — Grows on all media, but quickest and most characteristically on Loeffler' s blood serum : [beef-blood serum, 3 parts, 1 per cent glucose broth (meat infusion), 1 part. Put in tubes, slant, coagulate at 700 C, and sterilize at 570 C. for 1 hour on eight days.] On this medium colonies form in from 12 to 24 hours at 370 C, as small circular discs of opaque whitish colour, like candle- grease spots, and enlarging rapidly, outstrip any accom- panying colonies of streptococci. On agar, a similar growth occurs, but less quickly, and is closely resembled by that of Streptococcus pyogenes. In broth, a pellicle may form, and a turbidity which however soon settles to the bottom, leaving the fluid clear and producing a powdery deposit. It grows well in milk but does not clot it. Does not form indol. Ferments glucose, galactose, laevulose, maltose, and glycerin, but not mannite or saccharose. It reduces nitrate to nitrite. NON-SPORING BACILLI 247 Grows best at 370 C. ; growth at 220 C, but not at 20° C. Isolation. — Take sterile swab, rub on throat, then rub over serum in tube, and incubate for 18 to 24 hours at 370 C. Take platinum loop and rake all over surface of serum, and then make a film and stain with Loemer's methylene- blue or Neisser. Distinguish from Hoffmann's bacillus, which is shorter and plumper, does not produce acid in glucose, is non-pathogenic to guinea-pigs, and does not show granules with Neisser. (B. diphtherias at times does not give Neisser, and occasionally pseudo-B. diphtherias does stain with Neisser.) The only sure method of differentia- tion is toxicity of culture ; guinea-pig killed in 24 to 48 to 72 hours by injection of toxin of true B. diphtherias. In the serum-water medium of Hiss, plus 1 per cent of glucose, lasvulose, galactose, maltose, lactose, saccharose, mannite and dextrin, B. diphtherias produces acid in all except with mannite, lactose, and saccharose ; B. xerosis produces acid with all except mannite, lactose, and dextrin ; B. Hoffmanni does not produce acid with any. The saccharose and dextrin media therefore serve to differentiate : B. diphtheria, acid with dextrin, not with saccharose ; B. xerosis, acid with saccharose, not with dextrin ; B. Hoffmanni, acid with neither. In Vincent's angina, B. fusiformis is found : an anaerobe, Gram-negative, longish, and swollen in the middle. B. xerosis is found in the conjunctiva, and is pfactically identical with the B. diphtherias : it is non-pathogenic to animals ; produces no toxin ; does not form acid with glucose, but ferments saccharose. Habitat. — Mucous surfaces, mouth, throat, nose, con- junctiva, larynx, middle ear, vagina. Thermal Death-Point. — Forty-five minutes at 550 C, (moist heat) ; dry membrane, 1 hour at 980 C. Resistance. — Lives six to eight weeks on agar, five to six months on serum, twelve to fifteen months on dextrose serum. In dried membrane retains vitality for months. Pathogenicity. — Guinea-pigs die in from two to three days after subcutaneous injection of young broth culture. Nephritis and paralysis are observed, but the characteristic feature is enlarged and hasmorrhagic condition of the 248 PUBLIC HEALTH BACTERIOLOGY adrenals. Cats, dogs, and pigeons are very susceptible to mucus infection ; rats and mice are refractory. A guinea-pig killed by the injection of a virulent culture of diphtheria bacillus shows congestion of all the organs, especially severe in the suprarenals. Toxins. — Diphtheria toxin is an extracellular one, that is, it is soluble in the liquid media, and can be obtained separate from the bacillus by filtration through a Chamber- land tube. Toxin formation is best got in meat-infusion broth with added peptones, and rendered alkaline, after two to three weeks' growth at 37-5° C. A free supply of oxygen is important to secure the greatest toxin formation. The toxin is readily destroyed by bright light, by exposure in a liquid solution to 6o° C, or if in dry state to a temperature over 700 C. Sealed and kept in the dark and in the cold, it may be kept for long periods. It is believed to be closely allied to the albumoses. Antitoxin. — The mode of preparation and standardiza- tion is described under Antitoxic Sera (page 189). The value of antitoxin in the treatment of diphtheria is now well attested. The results are better the earlier the injec- tion, so that few deaths occur in those injected within twenty-four hours of the onset, the rate gradually rising until by the fifth day the effect is slight. The dose given is not proportionate to the age, but to the severity of the attack and the time that has elapsed before coming under treatment. 2000 to 8000 units should be given to children on these principles, and up to 50,000 units have been given in one case. In large doses, it is better to give the higher-potency sera, in which 10,000 units can be had in 10 c.c. of serum. In about one-third of the cases injected, serum sickness or serum disease is noted. The symptoms are : an erythematous rash and fever coming on in a week to ten days after the injection. At times general pains and even arthritis may be present. These symptoms are more likely to be produced by large doses of serum. But more alarming symptoms than these occur soon after the injection, as has been referred to under Anaphylaxis (page 212). On analysis these cases are found to occur in people who have not been previously injected, and in people who have had a previous injection NON-SPORING BACILLI 249 more than the incubation period of the serum disease (that is, ten to twelve days) before. On account of these facts, Goodall advises that prophylactic doses of diphtheria antitoxin should not be given to anyone without discrimina- tion, and never to a person the subject of asthma (see page 213) . In an actual attack of diphtheria in such a person the risk would have to be definitely considered, and a judgment come to on the relative dangers of the attack and the use of the antitoxin. In those who have had a previous dose, either for an attack or for prophylaxis, some time antecedent, one should be on the outlook for alarming symptoms if a second dose is being given. It is stated that so far no death has been recorded in these circumstances, which is to some extent reassuring. This class of case suggests the avoidance of prophylactic doses altogether, as if the person takes an attack more than ten days later, he is sensibilized to the now required injection. (See for further details, articles by Goodall in Public Health, January, 191 1, and in Encyclopedia Medica ; and by Currie, Journal of Hygiene, January, 1907. Also annotation in Lancet, 1911, vol. i, page 1654.) Antitoxic serum keeps well in a cool, dark place. Anderson has found that at 200 C. the average yearly loss of potency is 20 per cent ; at 150 C, it is about 10 per cent ; and at 50 C, it is only about 6 per cent. Dried, and kept at 50 C, its potency was practically unimpaired after 5-5 years. The addition of chloroform, tricresol, etc., to preserve, had no influence apparently on the rate of deterioration. 250 PUBLIC HEALTH BACTERIOLOGY BACILLUS MALLEI. The B. mallei is the bacillus of glanders, a disease of horses, mules, and asses. Horned cattle are quite immune, whilst goats and sheep are intermediate in susceptibility. Guinea-pigs and rabbits can be infected by inoculation, but rats are immune. It was first obtained in pure culture and accurately studied by Loeffler and Schuetz in 1882, and from the human subject by Weichselbaum in 1885. Description. — B. mallei is a medium-sized rod (3 to 4 micra X 0-5 to 0-75 micron), straight or slightly curved, usually with rounded ends. It is about the same length as the tubercle bacillus, but distinctly thicker. These bacilli show considerable variations in size, even in the same culture ; and this is characteristic. They are non-motile, non-flagellar, non-sporing, and Gram-negative. They usually appear as single bacilli ; on rare occasions short filamentous forms occur. Staining. — The bacilli stain rather easily, but are as easily decolorized. The best results are got by staining with a mordant present, and simply washing in water, and drying ; in the case of tissues, dehydrating by the aniline- oil method. Carbol-fuchsin and carbol-thionin-blue make good stains. Loefner's methylene-blue, followed by slight decolorization in weak acetic acid, and then fifteen minutes in saturated solution of tannic acid, wash, dry, and mount, gives good results. As a counter-stain, 1 per cent acid fuchsin for half a minute may be used. With methylene- blue, the bacilli stain irregularly ; granular, deeply staining areas alternating with unstained or faintly-stained portions. This has been ascribed to degeneration, and to preparation towards spore formation. It is probably a peculiarity of the cell protoplasm. Cultures. — Grows well on all media at 350 to 370 C., indifferent to moderate degrees of acid or alkali. Glycerin or glucose render the media even more favourable. On agar and on glycerin-agar : the growth is greyish white to yellow. On gelatin : growth is slow, and greyish-white ; no lique- faction takes place. NON-SPORING BACILLI 251 In broth : diffuse clouding ; later, a heavy, tough, slimy sediment forms, and the broth becomes brown. In milk : coagulation takes place slowly, with slight acid formation. On potato : the growth at 37 ° C. is characteristic. Growth is rapid and abundant, and in forty-eight hours forms a transparent layer over the whole surface, and of a yellowish tint, like clear honey. The growth gets darker and more opaque, and on the eighth day it is reddish-brown or chocolate in colour, and at the edge the potato is stained a greenish-yellow colour. Spirilla cholerae and Metchni- kovi, and B. pyocyaneus give similar cultures. Resistance. — Killed by one hour at 750 C. or two hours at 6o° C. In the dark, in sealed tubes and on artificial media, it may remain alive for years. In watering-troughs, it has been found after seventy days. Complete drying kills* it in a short time ; carbolic 1 per cent, in 30 min. ; corrosive sublimate o-i per cent, in 15 min. Pathogenicity. — Notable for horses, mules, asses, cats, dogs, guinea-pigs, rabbits, and field mice. Non-sus- ceptible animals are : cattle, pigs, birds, rats, house mice, and white mice. In the horse, the disease takes two forms. When affecting the superficial lymphatic glands, it is called "farcy ; " when affecting the nasal mucous membrane it is called " glanders," and is a much more serious disease. In glanders the course may be acute or chronic. In the acute- form, chill is followed by general high temperature, and in a few days the nasal mucous membrane is studded with nodules, there is profuse nasal discharge, and later, ulceration of the nodules and swelling of the corresponding glands, and these also tend to break down. Finally the lungs are involved, and death takes place in from one to four weeks. In the chronic form, the onset is more gradual, the nasal swelling being accompanied by subcutaneous swellings all over the body, some of which tend to break down and ulcerate. These swellings are the so-called " farcy buds," and may persist for years. In man, the onset is usually violent, w7ith fever and general symptoms; and most cases terminate fatally within two to three weeks, sometimes within a few days. The infection is usually by a wound, which is followed by 252 PUBLIC HEALTH BACTERIOLOGY lymphangitis, and then a general infection resembling a pyaemia. As in the horse, the nasal mucosa tends to become affected. At times the course is more chronic. In the horse, the infection is usually by the mucous membrane of the nose or mouth, by wounds, or at times by the alimentary canal. Toxin. — No soluble toxin is described, but a concentrated three-weeks' culture in glycerin broth, sterilized by heat and filtered, is used as a diagnostic agent under the name of mallein (fluid). Dry mallein has been prepared by filtering a broth culture, concentrating filtrate on a water- bath to one-tenth of its bulk, and precipitating with thirty times its bulk of alcohol. Mallein differs from many other bacterial extracts in being extremely resistant to heat and storage without loss of strength ; 1200 C. has no destructive effect on it. Diagnosis of Glanders. — Three tests are used, namely : (i) Guinea-pig inoculation ; (2) Mallein test ; (3) Agglu- tination test. 1. Inoculation of a Guinea-pig. — A male guinea-pig is injected intraperitoneally with fragments of the diseased tissue, scrapings from ulcers, or nasal discharge of the suspected animal. A positive reaction is shown by the testicles becom- ing red and swollen usually on the second or third day, due to inflammation of the tunica vaginalis. Severe general symptoms follow, and death occurs in twelve to fifteen days. Greyish nodules are found in the spleen and other organs. The test is not absolutely specific, but is useful when other tests are inapplicable. A culture on potato of the pus from the tunica vaginalis should be made. 2. Mallein Test. — A proper dose of mallein is infected subcutaneously into the breast or neck of the suspected animal. It is advised to inject a dose into a control animal. The temperature of the animal should be taken at least three times a day for one or two days before injection. The injection is made at 6.0 to 7.0 a.m., and the reaction will be at its height at or before 10 p.m. of the same day. The temperature is taken every two hours after the injection for at least eighteen hours. On the NON-SPORING BACILLI 253 succeeding day take the temperature at least three times. In a healthy animal free from glanders, a local swelling, not exceeding 3 inches in diameter, is produced at the seat of inoculation, and a rise of temperature not exceeding i° C. (i-8° F.) ; and both swelling and temperature have much subsided in twenty-four hours. In a horse suffering from glanders, there appears within a few hours a firm, hot, diffuse swelling, which reaches a maximum size in twenty- four hours, is intensely tender during that time, and lasts from three to nine days. The size of the swelling reaches at least 5 inches in diameter. The temperature rises in six to eight hours 1-5° to 2° C. (270 to 3-6° F.), reaching 1040 to 1060 F. The high temperature continues for eight to ten hours (maximum about ten to sixteen hours after injection), and then gradually falls, but is distinctly above normal on the following day. This reaction is specific. 3. Agglutination Test. — The macroscopic or sedimentation method in high dilutions (1-1000) is preferred. Normal horse serum may react in 1-500. Immunity. — An attack of glanders does not confer immunity. Artificial active immunization has been attempted but has so far failed. Nodules. — The nodules found in glanders show more leucocytic infiltration and less proliferative change towards formation of epitheloid cells than does tubercle. Prevention. — The Glanders or Faro* Order of 1907, issued by the Board of Agriculture, (i)(jLays down com- pulsory notification of actual or suspected disease ; (2) Empowers local authority to slaughter at once any diseased horse, ass, or mule ; (3) Enables local authority to test suspected animals with mallein, and deal with contacts. 25i PUBLIC HEALTH BACTERIOLOGY BACILLUS PESTIS. This plague bacillus belongs to a group of bacilli which are all highly pathogenic to the animal world, and produce in them a haemorrhagic septicaemia. The bacilli now usually classed in this group are : the bacillus of human plague, of swine plague, of chicken cholera, of septic pleuro- pneumonia in cattle, and of rabbit septicaemia. The group characteristics are : short, plump, non-motile bacilli ; non- flagellar ; non-sporing ; Gram-negative ; non-gelatin-liquefy- ing ; strongly aerobic, growing readily on simple media, easily stained but showing a marked tendency to stain more deeply at the ends than at the centre (bipolar staining). They are believed by some to be varieties of one organism. Plague is a specific, infective disease, caused by the B. pestis, and characterized by inflammation of the lymphatic glands (buboes), carbuncles, pneumonia, and often haemorrhages (Osier). In the past the plague has occurred in tremendous epidemics, and even to-day in India it proves a terrible scourge. The large and extremely fatal epidemic of pneumonic plague in China in 1910-11 shows that it still has very pathogenic powers for mankind. In the sixth century, in the reign of Justinian, Emperor of Rome, half the population of the Roman Empire perished of the disease. In the fourteenth century the " black death " overran Europe and destroyed 25,000,000, or about one-fourth of the population. In the seventeenth century it raged virulently, and in London alone, in 1665, about 70,000 people died. During the eighteenth and nineteenth centuries its ravages lessened. In 1893 an outbreak appeared at Hong-Kong, and since then the disease has occurred in many parts of the world, notably in India since 1896, in Egypt, in South Africa, and in several Mediterranean ports, and, after an absence from Great Britain of over two hundred years, it obtained a foothold in Glasgow in 1900. It reached New York quarantine station in 1899, and in San Francisco broke out in 1900, continuing until 1904. In Australia, cases appeared at Sydney and other ports. In the county of Suffolk, in England, an epidemic of rat plague, associated NON-SPORING BACILLI 255 with a limited outbreak of pneumonic plague in man, was (September, 1910) the occasion of considerable anxiety and of increased vigilance and action. In California the last case of plague was noted in 1909, but an epizootic of plague among squirrels, causing thousands of deaths among these animals, was only reported as quiescing in 191 1. In 1894, Kitasato and Yersin independently discovered the bacillus in large numbers in the buboes, cultivated it in pure growth, and reproduced the disease in susceptible animals by inoculation, and from them recovered the bacillus. The proof in the human subject was given later, when by an accidental infection a physician and a nurse died of plague. Description. — B. pestis is a short thick bacillus with well-rounded ends, thus appearing as a small oval rod, two to three times in length what it is in breadth (1-5 to 175 X 0-5 to 07). The bacilli appear singly, though at times in pairs, and in fluid cultures in chains. In young cultures they show marked variations in size, and less polar staining. In old cultures, involution forms appear, as swollen coccoid forms, or as longer club-shaped diphtheroid forms. In the tissues they are sometimes found to possess a capsule. They stain readily with all the usual aniline dyes, dilute aqueous fuchsin and methylene- blue having been mostly used, and these show the polar staining well. Special polar stains have been devised. Involution forms are developed more rapidly when NaCl is added to the medium, and " salt agar " containing 2 to 5 per cent of NaCl is used for diagnostic purposes, the bacilli showing the usual shape on plain agar, on salt agar exhibiting coccoid, root-shaped, large, globular, and sausage-shaped forms, when the higher percentage is used ; with the lower percentage, the most striking feature is a general enlargement of all the bacilli. Dr. R. M. Buchanan, city bacteriologist of Glasgow, in a Report to the Local Government Board for Scotland ("Thirteenth Annual Report of the L.G.B., Scotland, 1907," page 81), describes a new culture medium for B. pestis as follows : " Rat agar as a culture medium for B. pestis. The susceptibility of the rat to plague suggested the use of rat tissues 256 PUBLIC HEALTH BACTERIOLOGY instead of ox flesh in the preparation of a nutrient medium for the growth of Bacillus pestis. An extract is made from the carcases of rats deprived of skin, head, stomach, and intestines, and the further preparation of the medium is essentially the same as in that of ordinary agar, except that the extract is boiled for half an hour before straining. Rat agar has been used in the (city) laboratory for a number of years as a culture medium for Bacillus pestis, with most satisfactory results, growth taking place on this medium with much greater certainty, rapidity, and profusion than on glycerin agar. It is also noteworthy that on this medium the bacillus closely approximates to the form which it assumes in the body, and is sometimes much elongated. In this elongated form it may be difficult to recognize, so different is it from the familiar cocco-bacillary growth of glycerin agar." B. pestis is Gram-negative, non-gelatin-liquefying, non- motile, non-sporing, and non-indol forming. Cultures. — Grows readily and luxuriantly on all the meat-infusion media. The optimum temperature is 300 C, while below 200 C. and above 380 C. the growth is sparse and delayed. The best reaction is neutrality or slight alkalinity ; but acidity does not prevent growth. On agar : growth appears in twenty-four hours as minute colonies, whitish and compact in centre, and showing to hand lens a broad, irregular, indented, granular margin. Kept for a few days at room temperature, some colonies grow faster than others and become more opaque, almost suggesting a mixed growth (Muir and Ritchie). On gelatin : a similar growth occurs in two to three days. In stab, a white line of growth takes place along the needle track, and little or no surface growth. In broth : growth is slow, and usually forms a slightly granular or powdery deposit at the foot or sides of the tube or flask. If the surface is covered with " ghee " (in India, butter clarified by boiling, and thus converted into a kind of oil), delicate threads of growth extend from the surface downwards, the so-called " stalactite " growth, which however is not specific to the plague bacillus, nor is it shown by all races of the organism. To observe it, the culture must be kept absolutely at rest. NON-SPORING BACILLI 257 Milk is not coagulated. Litmus milk shows slight acid formation. Growth on potato and blood serum shows nothing of differential value. • In peptone water, no indol is formed. Resistance.— Readily killed by heat, like all non-sporing forms ; ten minutes at 650 C., or one hour at 580 C. Drying kills them in six to eight days ; artificial drying in four to five hours. May live in pus or sputum for eight to fourteen days. In a moist dark place, may retain viability for months or even years. Freezing has little effect, bacilli surviving a temperature below o° C. for forty days. Direct sunlight kills them in four to five hours ; carbolic 1 per cent in two hours, 5 per cent in ten minutes ; perchloride of mercury 1-1000 in ten minutes. Pathogenicity. — For man, very pathogenic. For animals, very marked for rats, mice, guinea-pigs, rabbits, and monkeys. In rats and guinea-pigs, the mere rubbing of plague bacilli into the skin will often produce the disease, and this fact is made use of in isolating the bacillus from material contaminated with other bacteria. Animal passage increases virulence ; growth on artificial media diminishes it, when prolonged. After subcutaneous injec- tion, a local inflammatory swelling follows ; then a swelling of the corresponding lymphatic glands ; thereafter a general infection. Mice usually die in one to three days, rats and guinea-pigs in two to five days, and rabbits in four to seven days. Post mortem, the chief changes are : the enlarged glands, congestion of the organs (sometimes with haemorrhages), and enlargement of the spleen. The bacilli are numerous in the lymphatic glands, usually in the spleen, and throughout the blood. The blood of a plague-stricken rat may contain as many as 100 million bacilli per c.c. Transmission. — Actual contact plays a very minor part in the transmission of the disease, as the virus is not given off by the skin. The chief modes of transmission are two in number, by : (1) Inoculation by biting insects (Limond, 1899), tne usual insect being the rat flea (Pulex cheopis) ; (2) Inhalation : this is the mode of spread of the pneu- monic form of plague. 1. By inoculation by biting we get the bubonic plague, 17 258 PUBLIC HEALTH BACTERIOLOGY the course of which corresponds exactly to that induced in animals by subcutaneous injection, as described above. An outbreak of bubonic plague is always preceded by an increased death-rate among rats, and that from a disease now known to be due to the B. pestis. The rat flea becomes infected by sucking the blood of the rat, and infects other rats, or it may be human beings. It is now believed that the flea does not inject the bacillus when biting, as no bacilli have been found in its biting apparatus. It is therefore surmised that the mode of infection is by the inoculation of the biting wound by the rubbing in of the excreta and vomit of the flea, both of which are highly charged with B. pestis. The proof that B. pestis multiplies in the stomach of the flea is held to follow from the fact that abundant bacilli may be found in it up to twelve days or longer. It has also been observed that in India plague does not maintain itself in epidemic form after the mean temperature has risen above 8o° to 85 ° F. (26-6° to 29-4° C). This has been found to be associated with a rapid disappearance of the bacilli from the alimentary canal of infected fleas during the prevalence of this higher tempera- ture. Transmission by inoculation must be extended to the infection of attendants on the sick and others, by the rubbing in of soiled linen, etc., and also from earth soiled by the excreta, vomit, and sputum of rats dead or dying of bubonic or pneumonic plague. Such transmission is urged by some as a likely one in the case of barefoot peoples, living in dwellings with earth floors and infested with rats. The transmission from rat to rat is believed to be by the flea, in which case the buboes are mainly cervical ; and by ingestion of rats dead of plague by other rats, when the buboes are mesenteric. The subject of transmission in this manner has been mainly studied in India. In Bombay there are two kinds of rats : (1) Mus decumanus, and (2) Mus rattus. Both kinds are infested by the same flea, the Pulex cheopis. The Mus decumanus is the large brown rat, and is the same species as that present in Suffolk, England, to-day. The Mus rattus is the black rat, and is identical with the English black rat. These two species of rats differ fundamentally in their habits. The Mus decumanus is a timid rat, which NON-SPORING BACILLI 259 avoids man as far as possible, and finds its food in sewers, ditches, fields, etc., rather than in inhabited houses. Mus rattus, on the other hand, is a domestic animal in India, and lives in close and intimate association with the home-life of the people. The Mus rattus, therefore, is chiefly responsible for the transmission of the disease to man ; while the Mus decumanus is of special importance in maintaining the disease from season to season. The Indian Plague Commission conclude that plague is a rat disease, having a regular periodicity, namely (a) an epizootic season, from December to May inclusive, and (b)t a non- epizootic season, from June to November inclusive. During the latter period there are few cases of plague in rats, fleas are scanty (this is given as a cause of the decrease in cases of plague in rats), and in some villages where the Mus rattus alone prevails, plague may actually die out each season. The Mus decumanus is more infested with fleas, and is thought to keep the infection going from season to season. A fresh epizootic first chiefly affects the Mus decumanus, then spreads to Mus rattus, and then to human beings. In this way are explained the outbreaks of plague in India, year after year since 1896, causing nearly a million deaths in 1904 among the natives, while the attendants and Europeans enjoy almost complete immunity, although both hospitals and camps abound with Pulex irritans. This is ascribed to the habits of this common flea of man, in that it rarely bites other creatures than man, and that in man with plague the blood is not so alive with bacilli as in the rat. The chances of infec- tion by Pulex irritans are from these causes enormously reduced. 2. By inhalation of the virus, causing the pneumonic form of the plague, which is very rapid and fatal. It is not understood what factors determine the appearance of the disease in this form, but once started it is extremely infectious. The symptoms are very similar to those of acute lobar pneumonia (although the pneumonia is mainly lobular in its distribution), with high fever, rapid respiration, and hemorrhagic sputa. The sputum contains the bacilli in enormous numbers, and almost in pure culture. In Egypt, the summer type is bubonic, and the winter type pneumonic. 260 PUBLIC HEALTH BACTERIOLOGY Cimex lectularius, or the common bed-bug, has lately been investigated as a carrier of plague bacilli. Enormous numbers can be found in the stomach of the bug after infection, and for four to five days. Two bugs were still alive eighty-three days after the feeding, and broth and agar cultures were obtained from their bodies. Inoculation of mice produced typical results, and the B. pestis was recovered. Other types of plague are described, which do not come under the above headings. These are : the ambulant form, in which the patient has a few days of fever, with swelling of the glands of the groin, and possibly suppuration. These cases are often found at the beginning of an epidemic, and are a source of great danger to the community, as the urine and faeces contain bacilli ; and the septicemic type, in which the patient succumbs to a virulent infection before the buboes appear. There are also cutaneous and intestinal types, the former showing petechias and subcutaneous haemorrhages ; the latter, diarrhoea and haemorrhages from the mucous membranes, and sometimes the features of enteric. In India, an analysis of n,6oo cases gave 77-65 per cent of bubonic type, 14-25 per cent of the septicaemic type, and 4-4 per cent of the pneumonic type. The mortality was highest in the pneumonic type (96-69 per cent), and almost as high in the septicaemic. In the bubonic form, out of 9,500 cases, 5,130 (54 per cent) showed the glands of the groin first affected, and usually on the third to the fifth day.* Resolution may occur, or suppuration, or in rare cases, gangrene. Suppuration is noted as a favourable feature. The appearance of petechiae, or " plague spots," or " tokens of the disease," gave to it in the middle ages the name of the " black death." Toxins. — The filtrate of a plague culture has a very slight toxic effect, but not capable of inducing immuniza- tion. Hence it is believed that little or no soluble toxin exists. Injection of dead bacilli produces distinctly toxic * The areas of skin surface which drain respectively into the glands of the groin, axilla, and neck, are as 5 : 1*8 : 1, and the number of primary buboes observed in hospital in these glands were as 5-8 : 1-3 : 1 respectively. NON-SPORING BACILLI 261 effects. These endotoxins are comparatively resistant to heat, being unaffected by exposure to 65 ° C. for one hour. By the injection of these endotoxins in suitable doses, a degree of immunity against living virulent bacilli is obtained. The serum of such immunized animals is found to confer a degree of protection on small animals. Immunization. — 1. Preventive inoculation. — Haffkine's method. Cultures are made in flasks, with oil drops on the surface. Stalactite growths form, and the flasks are shaken every few days to break those formed, and so induce fresh crops. The incubation temperature is 25 ° C, and six weeks' growth is allowed. Thereafter the culture is sterilized by heating for one hour at 650 C, and carbolic acid is added to make the bulk contain 0-5 per cent. The contents are well shaken to distribute the sediment, and then bottled for use, the fluid thus containing the dead bodies of bacilli as well as any toxins that may be in solution. It is administered subcutaneously, and usually in one dose of 5 c.c. The susceptibility is said to be reduced to one- fourth, and the mortality among those inoculated who take the disease is about one-half of that among the non-inoculated. Protection begins a few days after inocu- lation and lasts for several months. 2. Anti-plague Serum. — Yersin has prepared a serum from horses, by injecting dead bacilli into the sub- cutaneous tissues, then into the veins, and finally, living bacilli intravenously. After a time, blood is drawn off, and the serum preserved in the usual way ; 10 to 20 c.c. are injected daily. Some curative power has been observed. Serum Diagnosis. — Specific agglutinins appear in the blood of some patients, but the potency of the serum is not strong, and the test is not easily carried out, owing to the tendency of the bacilli to adhere in clumps preventing a satisfactory emulsion being got. Hence the macroscopic or sedimentation method is preferable. Cairns, in the Glasgow cases, found that the reaction appeared about a week after onset of the illness, increased until the sixth week, and then faded away ; being most marked in severe cases taking an early and favourable crisis, less in severe cases tending to a fatal issue, and feeble or absent in the mild cases. The best dilutions were 1-10 to 1-50. 262 PUBLIC HEALTH BACTERIOLOGY Methods of Diagnosis. — Bubonic. — (i) Prepare the skin over a bubo, and remove some juice by aspiration with a sterile hypodermic syringe, the needle of which is plunged into the bubo ; (2) Make smears, and cultures on agar and salt agar ; (3) Inoculate a guinea-pig with some of the material, by rubbing it in on the shaven skin with a glass spatula, or by subcutaneous injection. In bubonic plague, a diagnosis in many cases can be made by microscopic examination alone, as in no known condition other than plague do bacilli with the same morphological characters occur in such numbers in the lymphatic glands. Pneumonic. — (1) Microscopic examination of the sputum ; (2) Make cultures, inoculate a guinea-pig ; also a rat by smearing the sputum on its nasal mucous- membrane. In pneumonic plague, a positive diagnosis should not be given from microscopic examination alone, especially in a plague-free district, as bacilli morphologically resembling the plague organism may occur in the sputum in con- ditions other than plague. Post Mortem of Rat Dead of Plague. — Subcutaneous injection of the flaps of the abdominal wall is noted. Fluid in the pleural cavities, haemorrhagic oedema of the neck glands, a creamy mottled appearance of the liver, and a somewhat similar appearance of the spleen. The Indian investigators laid stress on an abundant, clear, pleural effusion. The neck glands are chiefly involved in the rat because the flea prefers the skin of the neck ; in California and Glasgow, however, the cervical bubo was not so commonly found as in India. In chronic rat plague, enlargement of the spleen, with the formation of nodules in it containing plague bacilli, was the usual finding. Prophylaxis. — Destruction of rats, by poisoning, trapping, ferreting, and virus. Separation of rats from mankind by better drainage, paving of yards, concrete floors, and general repairs. NON-SPORING BACILLI 263 Removal of rat food, by frequent scavenging and attention to the feeding of fowls, pigs, etc. Slaughter-houses require special attention. General campaign of cleanliness, avoidance of fatigue of body or mind, and temperance in all things. Haffkine's prophylactic inoculation, at least to all those on the staff or otherwise specially exposed to infection. Nursing. — Bubonic plague requires no special pre- cautions, as it is not infectious. The liberal use of iodoform as a dusting powder, on the person and clothing, is strongly recommended to prevent the attacks of fleas. Pneumonic plague is acutely infectious, hence both doctor and nurse should wear a cotton-wool respirator, as should also the attendants. Summary. — Plague is a rat disease. It is conveyed from rat to rat, and from rat to man, by the rat flea. The human flea is not involved to any extent in the matter. Bubonic plague is not an infectious disease, as this phrase is commonly understood. Pneumonic plague is most infectious, apart altogether from the question of fleas. Plague pneumonias breed true, i.e., give rise to other cases of pneumonic plague. The B. pestis blood-count is low in man, high in the rat. This affords an explana- tion of the high degree of infectivity of the rat flea as compared with the human flea. Insanitary conditions apart from the presence of rats play a secondary part. In Suffolk, Mus rattus is rare, Mus decumanus is common ; therefore close contact with plague-stricken rats is unlikely, and hence the small epidemic. (Pringle, " The Outbreak of Rat Plague in Suffolk," Public Health, January, 1911.) For an important article on the " Spread of .Plague," by C. J. Martin, and the subsequent discussion, see Brit. Med. Jour., 1911, vol. ii, p. 1249. Summary of the " Lancet " Reports on the Plague in China, 1910-1911. The outbreak of plague in China, beginning in Manchuria on October 12th, 1910, and extending rapidly until it had 264 PUBLIC HEALTH BACTERIOLOGY invaded widely separated districts, was notable in several important particulars. The epidemic was almost without exception one of primary pneumonic plague. The fatality was extremely high, few cases of recovery having been reported. The infectious nature of the malady was very great, and the virus was apparently carried by the sputum. The origin of the plague was not ascribed to rats, but to marmots, a species of squirrel living in burrows. The question of infection by fleas is here of minor importance, once the epidemic is started. Its relation to the origin of the epidemic has not been worked out. Once begun, the propagation was apparently by direct inhalation of the virus. The Chinese Government invited the other Governments to send representatives to an International Plague Conference, which began its sittings at Mukden on April 3rd, 191 1. The following statements are taken from the reports of the proceedings published in the Lancet from April 29th, 191 1, onwards, which include an exhaustive report by Dr. G. Douglas Gray, physician to H.B.M. Legation, Peking, the questions for discussion, and the Chairman's inaugural address. Origin. — It had been known for many years that in Eastern Siberia and Mongolia the marmot, or tarabagan (Russian), or han ta (Chinese), a variety of the squirrel tribe, of the rodent genus, frequently surfers from a fatal disease which may be transmitted to man and produce symptoms indistinguishable from bubonic and pneumonic plague. This animal is hunted for its fur, which is used to imitate sable and other furs. One of its favourite haunts is a mountain range in the north-west of Man- churia, and here large numbers of Chinese are employed trapping it during the summer months. It was among these trappe*rs that the present outbreak is held to have originated. The proof that the " tarabagan disease " in man, and plague, were one and the same disease, has not been given bacteriologically, and in 1905, 1906, and 1907, Russian scientific expeditions were sent to investi- gate and report. Dr. M. T. Schreiber concluded that : (1) Epizootics (a term applied to those animal diseases which behave as epidemics do in the human species) NON-SPORING BACILLI 265 undoubtedly do occur without human beings becoming infected ; (2) Field mice do not contract the disease from the tarabagan, though they have every chance of doing so, and are known to be susceptible to plague ; (3) Domestic animals also escape it, although dogs eat the flesh of the dead marmot. Dr. B. A. Barykin reported in 1909 the following facts : In 1906 the marmots around a settlement called Abogaitui showed a high mortality from spring to autumn, but the inhabitants were aware of the danger and avoided all contact with the sick animals, except a Cossack, who was in indifferent health and had a craving for the flesh of a tarabagan. He got some, fell ill with the symptoms of plague, and died in four days. Others became ill, and in all eight died with symptoms of pneumonic plague. Post mortems were held in two cases, and bacilli indistinguishable from the plague bacilli were found in the organs ; and mice injected with the splenic juice died in twenty-six hours with the typical appearances. This was in September, 1906. In the autumn of 1907, marmots were caught or killed by the party and examined for the presence of disease. In one of these animals showing no external signs of being ill save some degree of malnutrition, the spleen was found to be swollen -and congested, and contained large numbers of bacilli identical in morphology and culture with the plague bacilli. A fortnight later, twelve miles from where this animal was discovered, the men of an isolated Cossack family, hunting the marmots around them, killed one showing clear signs of illness. In spite of the counsel of the elder men the animal was skinned and the body was given to a girl of thirteen years to take to the fields. She dragged its body (said to be 15 lb. weight) after her through the grass, and returned barefoot over the same path. On the next day she fell ill, a bubo appeared in the left groin, and she died some days later with all the symptoms of plague. From the bubo, from a pustule on a finger, and from the spleen, bacilli indistinguishable from plague bacilli were isolated, and being injected into mice caused their death in eighteen hours, of septicaemia. Neither the body nor the skin of the animal was recovered. 266 PUBLIC HEALTH BACTERIOLOGY The rest of the family escaped. The same autumn a railway guard who had caught marmots, and a woman who had skinned them, both died of a disease resembling bubonic plague. In the guard, typical bacilli were found. A history of ten outbreaks in nine years in the district around one railway station was elicited. Various other localized outbreaks were reported, all tending to show that plague was not new to the districts where Mongolia, Manchuria, and Siberia adjoin. Spread. — Beginning then in the north-west borders of Manchuria among the marmot hunters, it was carried by these in travelling back to their homes, many of them having come from the Shantung province, south of Peking. In the third week of October, 1910, about 10,000 of these men had gathered in Manchourie and Khailar, stations on the Vladivostock railway, waiting to sell the skins they had gathered and to return to the south for the winter and the new year festival. Cases of illness occurred here, with symptoms of headache, fever, spitting of blood-coloured sputum, and followed by rapid death. Apparently in spite of the risks not many hunters die on the plains ; but when crowded into the poor hovels or inns of the market- towns, — where, in small badly ventilated rooms, twenty to forty may be found living, sleeping, and eating beside piles of raw pelts — the conditions for the encouragement of any epidemic disease are ideal. From these foci the infection spread by railway trains in which the hunters were carried to Harbin, then south to Chang-chun, thence to Mukden, on to Tientsin, and from there to their home villages in the Shantung province ; and also by foot travellers who struck across country through Kirin city {eighty miles from a railway) to Dalny, and thence by boat to Chef 00, a port in the Shantung province. All the way men were falling ill and dying, and the sick and dead were thus deposited at all the places passed en route. The infection reached Harbin on November 7th, and in three months there were 5,000 deaths out of a population of 30,000. Two factors seem to have contributed largely to the virulence of the epidemic in the Chinese city. First, the severe climatic conditions, the thermometer registering at times — 300 C. ( — 22° F., or 54 NON-SPORING BACILLI 267 degrees of frost), which prevented the people going out of doors. Secondly, the low, dark, dirty, and overcrowded houses which form the majority of the dwellings. Never- theless in Shuangcheng Fu, a finely planned city with wide streets, spacious compounds, and well-constructed houses, and with but little poverty, there were 1,500 deaths in seven weeks out of 60,000 inhabitants. Symptoms. — The incubation period in the majority of cases was five days. There were no marked prodromal symptoms. Often a man had a normal pulse and temperature on one day and was dead the next. The invasion was without rigor, with feelings of illness, weak- ness, and giddiness. A sudden onset with headache, then bloated face and suffused conjunctivae (septicaemic cyanosis), with temperature over 1030 F., and fast fluttering pulse, was usual. The respirations averaged thirty-five per minute. Coarse crepitant rales were noted all over the chest, but little or no impairment of resonance. These rales are due to marked oedema of the lungs in the late stages of the disease. In the earlier stages rales are rarely present even in serious cases, and then they are usually fine. Blood-stained sputum is often the first sign of illness in pneumonic cases. The signs of cardiac involve- ment are always marked in advanced cases : very rapid feeble running pulse, agonizing dyspnoea, galloping rhythm of the heart sounds, and sudden heart failure. Death occurs from the intoxication, with paralysis of the heart. Death resulted in attempts to move patients and where the patients sat up in bed for a few minutes to take nourish- ment. Labial herpes was not observed in any of the patients seen in hospital, which is a point noted before and interesting in comparison with acute lobar pneumonia, in which it frequently occurs. In the septicaemic form there may be a flow of blood from the nose or mouth shortly before death. No glandular enlargements were noted except once at Harbin, where there was a sub- maxillary bubo followed by secondary plague pneumonia and death. The bacteriological diagnosis was the only certain one, as the symptoms were so variable. Many were able to walk about until within a few hours of death, and up to that time declaring that they were quite well. 268 PUBLIC HEALTH BACTERIOLOGY Etiology and Pathology. — Dr. R. P. Strong and Dr. Teague performed twenty-five post-mortem examinations, and came to the following conclusions : That epidemic plague pneumonia results from inhalation, the primary point of infection being the bronchi. Through the bronchi the bacilli reach the lung tissue, and rapidly multiplying there they produce pneumonic changes of the lobular type and later more general lobar involvement. The blood becomes quickly infected and a true bacteriaemia results in every case. Secondary pathological changes occur, especially in the spleen, bronchial glands, heart, blood-vessels, kidneys, and liver. The fact that the bronchial glands at the bifurcation of the trachea are always much more affected than any of the other lymphatics, argues against the theory that epidemic plague pneumonia is primarily a septicaemia with secondary involvement of the lungs. Moreover, in the earliest stages of the disease, the blood may be free of plague bacilli. The condition observed in the trachea and bronchi in epidemic plague pneumonia is pathognomic of this condition alone. The throat and larynx may show characteristic appearances at times. The tonsils may become secondarily infected like other lymph follicles, but the duration of the disease is too short to allow of this as a rule. Primary infection by tonsil can occur, with enlarge- ment of the glands of the neck early in the disease. The oesophagus was found normal in every case, which argues against primary intestinal plague infection, since plague bacilli must have been repeatedly swallowed in sputum and bronchial secretion in many of the cases. Dr. Koulecha, who had dissected twenty-eight plague corpses, read a communication in which he differed in toto from the above in regard to the mode of infection. From the necropsies and microscopical examination of the tissues he concludes that pneumonic plague is a septicaemic disease, in which an overflooding of the blood and the lymphatic system with bacilli could be observed. Infectious matter entered the mouth, affecting en route the tonsils, mucous membrane of the trachea, bronchi, and neighbouring lymphatic glands ; and from these the bacilli passed into the blood. The lungs were apparently affected secondarily from the blood, which he inferred from NON-SPORING BACILLI 269 the great accumulation of the plague bacilli in the peri- vascular spaces. On this view, pneumonic plague is a lobar pleuro-pneumonia of hematogenous origin, and should be classed with croupous pneumonia. Dr. Fujinami confirmed these findings. Professor Zabolotny observed that it had not been sufficiently proved how many were of direct pulmonary origin and how many were of hemato- genous origin. Rats and Fleas. — Professor Kitasato reported that of 30,000 rats examined in South Manchuria, 6 per cent were Mus rattus, but none of the 30,000 showed plague infection. In North China, of 3,000 rats examined living by Dr Andrew, all were Mus decumanus ; he had never found Mus rattus. He had noted a seasonal flea prevalence, highest in September, October, and November. The only species noted was Pulex cheopis. Dr. Petrie found both Pulex cheopis and Ceratophyllus unisus among fleas examined at Mukden. Dr. Petrie also examined twelve tarabagans sent direct from Manchuria to Mukden. On these he found thirty-five fleas, twelve being on one alone. (Ticks were also observed on them.) The fleas found were unusually large, and on superficial examination appeared to belong to the genus Histricopsylla (eyeless, truncated head, comb on the inferior border of head, thorax, and part of abdomen, numerous long hairs over the body). In an epidemic of plague in Tongshan, in 1908, bubonic cases predominated at the first, but the proportion of septicemic and pneumonic gradually increased. Of the rats examined, 1 per cent were found to be affected. Some rats which remained well for days without any sign of glandular enlargement during life, were found post mortem to have Bacillus pestis in the splenic blood, and were evidently immune to infection, at least for the time being. Rats trapped in the mines in the neighbourhood were found to be less readily infected experimentally than those found above ground. The death-rate in the Tongshan epidemic was 800 out of 1000 cases. Diagnosis. — Sputum is scanty at first, and the bacilli are difficult to find in it. Later, it is almost a pure culture. Professor Zabolotny advised Gram's method for staining 270 PUBLIC HEALTH BACTERIOLOGY the sputum. He had often noted mixed infections, and a Gram-negative bi-polar-staining bacillus was seen at times, which was not the plague bacillus. It required further study. Involution forms bore no relation to virulence. Blood cultures could usually be got forty-eight hours before death. At least i c.c. of blood must be used for the test. In this way an earlier diagnosis could be made than by the sputum. In recovered bubonic cases, agglu- tination could be got in the second and third weeks with dilutions of 1-25 and 1-50. Agglutination and fixation of the complement experiments were of little value in pneumonic plague. Dr. Broquet advised the use of the following solution for the conservation of suspicious plague organs for future study or transmission to a laboratory at a distance : neutral glycerin, 20 c.c, calcium carbonate, 2 grm., distilled water, 80 c.c. Mix at 300 C., and immerse tissues. Vaccination. — Protective vaccination with attenuated plague bacilli was advocated by Dr. Strong. Professor Galeotti advised the vaccine of Lustig and Galeotti for these reasons : (1) The toxin (Galeotti holds that it is an endotoxin and non-soluble ; Zabolotny holds that it is a soluble toxin), and no other substance, is used ; (2) It can be dried and standardized ; (3) The plague nucleo-proteid can be stored in a sterile condition. The general experience was that no form of vaccination gave much protection against pneumonic plague. Serum Therapy. — Dr. Martini advised passive immuniza- tion for all those exposed to infection, such as doctors and nurses, etc. Small doses were useless ; 100 c.c. at least must be used, and repeated soon. As regarded the present epidemic, he thought the protective value was small. Professor Zabolotny reported that he had used up to 1 litre of serum without success, only prolonging the illness. Dr. Paul Haffkine had seen protective effect after large doses, but this did not last more than five days. Quarantine. — The use of railway cars for this purpose was a new feature. This gives a segregation camp divided into small units, each completely isolated from the other, easy of disinfection, easy to supervise and for the detection of onset of sickness. There were 3000 suspects thus NON-SPORING BACILLI 271 isolated at Harbin, the railway cars being drawn into specially constructed sidings. Disposal of the Dead. — With the ground frozen at — 40 ° C, ordinary burial of such large numbers of bodies was not easy. In spite of Chinese traditions, the Government gave orders for the cremation of the bodies of those dead of plague. This was carried out thus at Harbin : A pit 20 feet square by 10 feet deep was made by blasting with dynamite. When bodies were in coffins, these sufficed to aid the burning ; but if they were not, four pieces of round wood 4 inches in diameter by 2 feet long, were added per body. The pit held about 500 bodies, and into it kerosene was pumped from a fire-engine ; about 10 gallons per 100 bodies. On being set alight the mass burned fiercely and rapidly, and little but ashes remained. End of Epidemic. — The epidemic seemed to die out (April, 1911) when the temperature rose to — 200 C. ( — 40 F.). The total mortality was over 40,000. Summary of Conclusions. — The disease spread by direct infection from man to man. Whatever may have been its primary origin, there is no evidence that a concurrent epizootic in rodents played any part in its wide dissemina- tion. From Russian sources reports of an epizootic disease among tarabagans have been received, and it is not unlikely that this is plague, but the bacteriological proof is not yet complete. The infection was introduced into towns and villages by persons actually suffering from, or by those in the incubation stage of, the disease. There has been no positive epidemiological evidence to show that the disease has been spread by clothing, merchandise, or other inanimate objects. So far as can be ascertained the only infective agent in the epidemic has been the sputum of the plague patient. In the majority of the cases the disease has been contracted by the inhalation of plague bacilli in droplets of sputum, causing infection of the lower portion of the trachea and of the bronchi. In infection by inhalation the risk to the person exposed bears a direct relation to his proximity to the patient and to the duration of the exposure. In view of this, masks and goggles should be worn by all who come in 272 PUBLIC HEALTH BACTERIOLOGY contact with cases of the disease or suspected cases. The best form of mask is a three-tailed gauze bandage with a pad of cotton-wool. It should be destroyed or dis- infected after each exposure to infection. The epidemic was, almost without exception, one of primary pneumonic plague, with an incubation period of from two to five days. An increased temperature and pulse-rate are usually the earliest signs observable, but a diagnosis cannot be made until the organisms are recognized in the characteristic blood-stained sputum. An accurate diagnosis can only be made by a bacterio- logical examination of the sputum to exclude pneumonic infection due to other micro-organisms. Since the evidence points to the conclusion that in the epidemic all the cases became septicemic, an examination of the blood micro- scopically or culturally may be a valuable aid in diagnosis. The physical signs of lung involvement are too indefinite and appear too late in the course of the disease to be of diagnostic value, and even in cases in which the condition of the patient is grave they may be very slight. The fatality has been^ extremely high, scarcely any recovering. The general experience has been that no method of treatment has been of avail in saving life, but that the serum treatment seems in a few instances to have prolonged it. The decline of the epidemic has not been due to any loss of virulence of the bacillus, but probably to the preventive measures which were enforced either in accordance with scientific methods or by the efforts of the people to protect themselves. The statistics of prophylactic inoculation collected during the past epidemic do not allow of any definite conclusion being formed about their value in plague pneumonia ; but in bubonic plague it was argued that some degree of protection is conferred by the use of vaccines. Further experiments in animals are recom- mended, in reference to securing immunity against pneumonic plague infection. {Lancet, 191 1, vol. I, pp. 1614-16.) NON-SPORING BACILLI 273 THE TUBERCLE BACILLUS, This bacillus is the cause of tuberculosis, an infective disease, characterized by lesions which are nodular bodies called " tubercles," or by diffuse infiltrations of tuberculous tissue, which may undergo caseation and finally ulcerate, or may become sclerosed, and in some cases calcified. The infectious nature of tuberculous material was for long suspected, was proved by Villemin in 1865, and by Armanni and Cohnheim and Salomonsen from 1870 to 1880. Baumgarten first described the bacillus in sections, but Koch first established its causal nature on a solid basis by : (1) Demonstrating the presence of the tubercle bacillus in a great variety of tissues and organs ; (2) Pre- paring pure cultures of the organism from these ; (3) Producing the disease by the inoculation of the bacillus derived from pure culture ; and (4) Recovering the same organism from the diseased parts of inoculated animals. These four articles are known as Koch's postulates, and all of them have to be satisfied before an organism can be absolutely proved as the cause of a specific disease. At present, for quite a number of bacteria, some of these postulates have yet to be fulfilled as regards mankind. Description. — Bacillus tuberculosis is a slender rod, often slightly curved, measuring 2-5 to 3-5 micra long by 0-3 micron in width. They are of uniform thickness, or may show slight swelling at the ends or even in their length. They may lie singly in the tissues or sputum, but often in small heaps or masses. In cultures, a remarkable fila- mentous growth has been repeatedly observed. In the sputum, long branching hypha-like filaments, sometimes with swollen ends, have been found. These are looked upon by some bacteriologists as involution forms, but many regard them as evidence of the relationship of the tubercle bacillus to the hyphomycetes. A capsular or enveloping substance is produced by the bacillus, more by the human form than by the bovine, and more the longer the growth on blood serum. When stained, the bacillus often appears beaded, the unstained spaces being regarded as vacuoles by some and as spores by others. Inasmuch as the bacilli 18 274 PUBLIC HEALTH BACTERIOLOGY showing these have no increased resistance against heat and disinfectants, the spore interpretation is probably incorrect. The beads or highly stained portions have likewise been called spores, but the whole matter is at present unsettled. The tubercle bacillus stains imperfectly or not at all with ordinary watery aniline dyes, and only after long exposure or heating, or more quickly if a mordant is used. Once stained, the bacilli retain the dye tenaciously, in spite of treatment with alcohol or strong acids, and for the latter reason they are spoken of as " acid-fast " bacilli (acid- proof would be a better term). This feature seems to be due to the presence of fatty substances in the cell, and has furnished the basis for differential staining methods. The fatty substances are really wax-like in nature, and are soluble in alcohol + ether. B. tuberculosis is non-motile, non-nagellar, non-sporing, does not grow on gelatin, and is Gram -positive. Recently varieties have been described which are not acid-fast, but are stained by Gram's method prolonged, and these forms are stated to be present in old tuberculous lesions, where the ordinary form is not found and yet the material is virulent. These are, (i) A fine bacillary form, often showing granules, and (2) Free granules. Much also found that when acid-fast forms were added to milk (sterilized) and incubated, the acid-fast forms disappeared, and yet when the milk was injected into an animal, tuber- culosis was produced and in the lesions acid-fast bacilli were demonstrable. If these statements are conclusively proved, they are of the first importance. Chemical Analysis of Tubercle Bacilli. — Water 85-9 per cent, solids 14-1 per cent. The ash shows 55 per cent of P205, 12-6 per cent of CaO, 11-5 per cent of Mg, 13-6 per cent of Na20, 6.3 per cent of K20. Cultures. — The bacillus is not easily cultivated ; growth fails, or is slow or scanty ; fails on agar and gelatin entirely, but on glycerin agar (3 to 6 per cent) and in glycerin broth (6 per cent) growth takes place in ten to fifteen days, becoming visible first as dry white spots, the extension and fusion of which form a dull, whitish, wrinkled pellicle or layer. These media are suitable for subcultures ; for NON-SPORING BACILLI 275 growth direct from the tissues, blood serum and Dorset's egg media are commonly used. On blood serum, at 37-5° C, growth appears in ten to fourteen days as minute spots, rather irregular, raised above the surface, and comparable to small dry scales. The culture is got by inoculating with a sterile platinum loop from foci in a tuberculous gland ; or by planting an actual piece of tissue on to the surface of the serum ; or by inoculating with tubercle bacilli, derived by the anti-formin method (see later) from sputum or digested tissue. On Dorset's egg media, vigorous growth takes place resembling that on blood serum. To make the medium : take the whole contents of four eggs, beat well, add 25 c.c. of water, and mix thoroughly ; filter through muslin to remove air bells ; fill into tubes, and heat these in sloped position for four hours at 700 C. They are now ready ; but before inoculation, two drops of steri- lized water are placed on the surface of the medium. When inoculating, the material is well rubbed over the surface, the plug is replaced, and is sealed over with a few drops of paraffin, and the tube incubated in the sloped position. On glycerin potato, growth takes place, and on other purely vegetable media. Optimum Conditions. — B. tuberculosis is markedly aerobic. It grows at human blood-heat, 370 to 380 C. The usual range is 280 to 420 C. but it can be acclimatized to grow at 220 to 23 ° C. In fluid media it is killed in fifteen to twenty minutes at 6o° C, in five minutes at 8o° C, and in one to two minutes at 900 C. It can resist dry heat at ioo° C. for one hour. Simple drying is not efficient, as still virulent forms have been found in dried tuberculous sputum after two months. Similarly, putrefaction of sputa or tissue does not destroy the bacilli readily, for they have been found aliyo under such conditions after three weeks. Gastric juice failed to kill them in six hours, and several three- hour spells of freezing at — 30 C. had little effect. Direct sunlight rapidly kills them ; 5 per cent carbolic kills them in a few minutes, but if, as in sputum, they are protected by mucus, complete disinfection takes five to six hours. 276 PUBLIC HEALTH BACTERIOLOGY Perchloride of mercury is not very efficient, from the albuminate formed. The comparatively high powers of resistance (for an organism not admitted to have spores) are attributed to the protective qualities of the waxy cell membrane. The conclusion then is, that moist heat at ioo° C. will kill the bacilli in fluids and tissues, provided time is given for penetration of bulky materials. In Germany, tuberculous ox flesh is thus sterilized (four hours boiling) and then allowed to be sold. Pathogenicity. — For man is great : 10 per cent of all the deaths in Great Britain and in the United States of America are due to tuberculosis of one kind or another. This of course represents only a portion of the incidence of tuber- culosis, as many people are attacked and succeed in throw- ing off the infection. In fact it may be said that the fatal attack of tuberculosis is in many cases only the last of a long series of attacks more or less successfully repulsed. The commonest type in man is phthisis pulmonale s, then affections of lymphatic glands, bones and joints, and serous membranes. In America, 22 per cent of the deaths among the North American Indians, and 16 per cent among the negroes, are due to tuberculosis. For animals, the pathogenicity varies, but most are vulnerable. The question is at present clouded by the statement that various types of tubercle bacilli exist, such as the human type, the bovine type, and the avian type, and that these types vary in their power over the different animal species. Ignoring for the moment the different types, tuberculosis is mostly found in cattle and pigs. " Dogs and cats occasionally suffer, and monkeys and apes (immune when in the wild state) are very subject to it and mostly die of it in captivity. Horses are rarely attacked, and sheep are practically immune. Of all the home cattle slaughtered in the Glasgow market in 1910 (67,849), 12-98 per cent showed lesions of tuberculosis, and of 39,724 swine, 6* 12 per cent ; while out of 307,784 sheep not one snowed tuberculous lesions. The rate among cattle is excessive from the large proportion of milch cows included, some of which are imported into the city and are kept in byres NON-SPORING BACILLI 277 until sent to be slaughtered. Apart from that, however, the figures suggest the influence of open-air life in lowering the susceptibility of the sheep and the chances of infection. B. tuberculosis of the human type is pathogenic for guinea-pigs, less so for rabbits, and still less so for dogs. B. tuberculosis of the bovine type is very pathogenic for guinea-pigs, killing them more quickly and producing more extensive lesions than the human type ; while intravenous injection in rabbits causes an acute tuber- culosis with death in from two to five weeks ; the human type causing a mild, slow disease, usually lasting for six months, and at times failing to kill the rabbit. This is the readiest method of distinguishing these two types. The human type is found in the majority of human infections (46 out of 60 investigated), never in bovine tuberculosis. The bovine type is found always in bovine tuberculosis, and in a considerable number of cases of human tuber- culosis (14 out of 60 sb 23 per cent), and in all the latter but one, the lesions were of the cervical lymphatic glands, or were lesions of primary abdominal tuberculosis, that is, were lesions which might fairly be attributed to feeding or alimentation. Moreover they were mostly in children, so that the deduction is made that bovine tuberculosis is transmissible to man, the milk of tuberculous cows being the usual vehicle. Bovine tubercle bacilli, in cultures, are shorter, thicker, and more regular in size than the human type, and their growth on the various media is scantier. They are more pathogenic to cattle and swine, and less so to man, which may explain the chronicity of abdominal and cervical glandular tuberculosis. A recent research by Park and Krumwiede confirms the results of previous investigations. They state that com- parative luxuriance or sparseness of growth is in most cases absolutelv to be relied on for differentiation. 278 PUBLIC HEALTH BACTERIOLOGY Their results may be summarized thus : — Form of Tuberculosis Pulmonary Cer\ •ICAL Abdominal Type of bacillus found Human Bovine Human Bovine Human Bovine Ages of persons exam- ined : — 1 6 years and "upwards 5 years and up to 1 6 Under 5 years ** 290 3 7 r tji I o1 O 13 9 I 12 8 14 6 6 3 6 IO Totals 300 I 36 21 26 19 The results in 606 cases of various kinds of tuberculosis, investigated by different workers, are tabulated thus (three cases showed both types) : — Total Human Type Bovine Type Adults (16 years and upwards) Children (5 to 16 yrs. inclusive) Children (under 5 years) 389 78 136 381 - 98 % 54 = 69 % 99 * 72 % 37 • 2% 30 % 27% Totals 603 534 % The bacillus of avian tuberculosis shows a more luxuriant and moister growth than the human type, and grows at 43-5° C, is very pathogenic to rabbits, scarcely pathogenic to guinea-pigs, and not at all to dogs, even intravenously, whereas the human type produces an acute infection. Morphologically it is almost identical with the human type, and stains similarly. It is found in lesions in fowls and pigeons and some other bird species. Fowls fed on human tubercle bacilli are not found to become tuberculous. The identity of the two types is said to be established by the experiments of Nocard, who rendered mammalian tubercle bacilli pathogenic for fowls by keeping them in the peritoneal cavities of hens in collodion sacs for six months. Con- versely, prolonged cultivation and passage of the avian type through the mammalian body are said to cause them to approach closely to the mammalian type. The bacillus of a tubercle -like disease in a carp has NON-SPORING BACILLI 279 been called the bacillus of fish tuberculosis. It grows luxuriantly at room temperature (200 C), and does not grow at 370 C, its range being 15 ° to 300 C. It is non-pathogenic for mammals, but kills frogs in a month. It shows some degree of acid-fastness. Portals of Entry. — Inheritance, ingestion, inhalation, and inoculation. Cobbett recently traverses the theories of Behring, Calmette, and Guerin, that the portal of entry of the tubercle bacilli, even in the pulmonary form, is via the alimentary tract and thence via the lymphatics to the lungs. He concludes that the .intestine is not a common port of entry for the tubercle bacilli which cause phthisis. (Cobbett, " Portals of Entry of the Tubercle Bacillus," Jour, of Path, and Bad., vol. xiv., No. 4, 1910, p. 563). Leonard Findlay reaches the same conclusion from experiments on the production of pulmonary anthracosis in rabbits and guinea-pigs (Findlay, " The Origin of Pul- monary Anthracosis, an Experimental Study." Zeitschrift filr Kinder heilkunde, 1911, vol. ii., part 2 (June), p. 293. B.M.J., 1911, vol. ii., p. 1278.) Feeding experiments undertaken for the Royal Commission on Tuberculosis gave results more in consonance with Calmette' s theories (see resume on page 297 ; details in Appendix to Filial Report, vol. i., pp. 48 and 52). Toxins. — The tubercle bacillus secretes no soluble toxin or only in very small amount. The chief toxic principles are endotoxins or bacterial proteins. Dead bacilli will, if inoculated in sufficient numbers, produce tubercle-like nodules, in which giant cells and occasionally caseation are present. These results are obtained with intravenous and intraperitoneal injections, whereas subcutaneous injection produces a sterile abscess (cold abscess). The hope of producing an active immunity led Koch to employ various means to extract from dead and living bacilli the complex bodies bound in them. 1. Original Tuberculin, T.A. (Koch 1890-91). — Tubercle bacilli are grown in 5 per cent glycerin broth for six to eight weeks. The entire culture is then heated on the water-bath at 8o° C. until reduced to one-tenth of its original bulk. It is then filtered through sterile filter- paper or through porcelain filters. The filtrate is a thick, 280 PUBLIC HEALTH BACTERIOLOGY brown, syrupy liquid,, containing the products of growth in the culture medium and a 50 per cent extract of the bodies of the bacilli by glycerin ; all in so far as these are indestructible by heat. Stored in a cool dark place it keeps indefinitely, the glycerin acting as a preservative. The dose of this preparation used in cattle is 0-25 c.c, but the stock is usually diluted with 0-5 per cent carbolic water four times, making the dose 2 c.c. Such a dose in a healthy man causes in three to four hours malaise, tendency to cough, laboured breathing, and moderate pyrexia, all passing off in twenty-four hours. In a man suffering from tuberculosis, such a dose would give rise to such an excessive reaction as probably to cause death. Even o-oi c.c. causes all the above symptoms in an aggravated degree, together with marked inflammatory reaction around any tuberculous focus, resulting in necrosis but not killing the bacilli. This is now known as the " tuber- culin reaction," and is much used in veterinary practice. It is also used in clinical work for help in diagnosis of obscure affections suspected to be tuberculous. i. Subcutaneously : Use diluted tuberculin, 1-1000, so that 1 c.c. = 1 mgr. of tuberculin. Koch in 1890 used 1 «mgr., but now most clinicians begin with o-i mgr. or 0-2 mgr. If no reaction occurs, after three to four days give 1 mgr. If again no reaction follows, wait three to four days and try 5 mgr., and finally, if still negative, 10 mgr., but no more. If still no reaction, the person may be considered tubercle-free. Take temperature regularly before and after test. ii. Cutaneous test (von Pirquet, 1907). For this test von Pirquet first suggested a 25 per cent solution of " old tuberculin," but the latter is now used undiluted. The patient's skin on the flexor surface of the forearm is sterilized. Two separate drops of the tuberculin are placed on the skin, 2 to 4 in. apart. With a small metal bore the skin is scarified at a point midway between the two drops, and then that is covered by the drops. Within twenty-four to forty-eight hours in tuberculous patients redness round the points, a papule over the scarified surface, and minute vesicles all appear. The control area shows a slight traumatic redness which soon passes off. In a NON-SPORING BACILLI 281 negative result all three areas show this slight reaction. Intermediate reactions, in all degrees, are observed. 70 per cent of adults give a positive result, probably from healed tuberculosis. iii. Inunction of lanolin, containing 50 per cent of tuberculin (Moro's test). iv. Ophthalmo-tuberculin reaction (1907, Wolff-Eisner ; Calmette). For this test, "old tuberculin" is treated with 95 per cent alcohol, and the precipitate dissolved in water, and re-precipitated and dissolved several times, being finally made up to a 1 per cent watery solution. One drop of this is instilled into the conjunctival sac and allowed to spread all over it. A sharp congestion of both the ocular and palpebral conjunctivae results in six to ten hours in the case of a positive reaction, and passes off in twenty-four to thirty-six hours. In children, half the dose is used and the acme is reached sooner. In several cases the results of this test have been disastrous, the excessive reaction leading to destruction of tissue and blindness in the eye used for the test. 2. New Tuberculin (Koch, 1897) — Was introduced to pro- duce anti-bacterial immunity. It was prepared thus : bacilli from young virulent cultures were ground in an agate mill, washed with distilled water, and centrifugalized. The clear fluid was decanted and called tuberculin-O. The deposit was dried, ground, and treated as before ; and this was repeated several times until all the residue went into emulsion ; the resulting fluids mixed together are tuberculin-R. Tuberculin-0 gives no precipitate with glycerin added to make a 50 per cent solution. Tuber- culin-R does precipitate in 50 per cent glycerin. Tuber- culin-0 gives a reaction on injection more readily than tuberculin-R. 3. Bacillary Emulsion (Koch, 1901). — This is a 1 per cent emulsion of pulverized bacilli in distilled water, allowed to sediment for several days, and then the super- natant liquid is mixed with an equal bulk of glycerin, making the whole 50 per cent. 1 c.c. = 5 mgr. solids. It resembles a mixture of tuberculin-0 and tuberculin-R. 4. Bouillon Filtre (Denys, 1905). — A culture of tubercle bacilli in 5 per cent glycerin broth, filtered through 282 PUBLIC HEALTH BACTERIOLOGY Chamberland filters and not heated, but 0-25 per cent phenol added to preserve. Corresponds to " old tuberculin," unconcentrated and unheated, and therefore supposed by Denys to contain important soluble but thermolabile substances. Therapeutic uses of the Tuberculins. — (So-called Vaccine Therapy.) — For these purposes, tuberculin-R and bacillary emulsion are mostly used in doses beginning at o-ooi mgr. (yyVo)' and gradually increased so long as the dose causes no greater disturbance of temperature than 0-5° F. In Wright's method of treatment the initial dose is usually ToVo mgr-> and is rarely increased beyond ToVtt mgr. The dose is administered after determination of the opsonic index of the patient, and subsequent doses are only administered during the positive phase of the reaction to the previous dose. To determine this, repeated observations of the opsonic index have to be made. As a result of experience gained by a large number of observations, some authorities advise the use of smaller doses, 5l[iM mgr. every ten days, gradually increased to 40*00 mgr. in six months' time, without opsonic index estimation, but simply watching the clinical symptoms. In the serum of patients so treated there is evidence of the formation of bodies antagonistic to tuberculin, of the nature of immune-bodies, precipitins, and opsonins. Antituberculous Sera. — Maragliano's Serum. — Made by him by immunizing dogs, asses, and horses, by injecting a mixture of the filtrate of an unheated broth culture (1 part) and an aqueous bacillary extract at ioo° C. (3 parts). The animal is bled after four to six months. Of the serum, 2 c.c. are injected subcutaneously every two days. Improvement has been noted in non-febrile cases. The serum is capable of protecting an otherwise healthy animal against a fatal dose of tuberculin. Marmorek's Serum. — Marmorek believes the tubercle bacillus does not produce in ordinary media the same toxins that it does in the body, where it has to resist the antagonism of the body cells. To combat this he first grows the bacilli on a leucotoxic serum (produced by inoculating calves with guinea-pig leucocytes), and then on a medium containing liver extract, the liver being NON-SPORING BACILLI 283 regarded as the most antituberculous tissue in the body. He thereafter uses these bacilli (which he states yield no tuberculin) to immunize animals, and for the serum produced he claims high curative powers. Tuberculin Tests applied to Cattle. — In cattle, tuberculosis may be present without any very apparent symptoms until an advanced stage is reached. Routine examination of herds by the tuberculin test has therefore become one of the necessary measures in public sanitation, in order that the milk of tuberculous cows may be excluded from consumption, and that such cows may be eliminated from herds (Bang's System). Mohler states that an accurate diagnosis is established in 97 per cent of the cases. Stall the animal and take the temperature in the rectum every two hours from 6 a.m. until midnight. Make the injection then (subcutaneously). Begin to take the tem- perature at 6 a.m. and continue as on the preceding day. The dose is usually 0-25 c.c. of old tuberculin. A positive reaction consists in febrile and constitutional signs, with marked congestion around any focus of tuberculosis. The febrile reaction begins six to ten hours after injection, reaches its height in nine to fifteen hours, and declines to normal in eighteen to twenty-six hours. A rise of 20 F. or more above the maximum of the previous day should be regarded as a positive reaction. In a doubtful case, repeat in four to six weeks. (Normal rectal tempera- ture, ioo° to 1020 F. ; normal pulse, 45 to^55 beats per minute. Methods of Detection. — 1. Microscopic. — In sputum: select a yellowish piece, make a film, and stain by acid-fast method. If not found, make solution of sputum by gradual addition of NaOH solution, boiling the while ; then sediment or centrifuge. Or mix in 5 per cent carbolic or 2 per cent lysol, and stand ; gradual solution, and tubercle bacilli precipitated. Or add antiformin (or a mixture of equal parts of solutions of NaCIO and NaOH, each 7-5 per cent) ; dissolves in a few minutes. If a 20 per cent dilution of antiformin is used, the tubercle bacilli are not damaged, and all the other bacteria are killed. Centrifuge or sediment ; wash twice with normal salt solution, and sediment can then be used to make culture, 284 PUBLIC HEALTH BACTERIOLOGY or inoculate guinea-pig. In urine : sediment or centri- fuge, make films, and examine. To avoid smegma bacilli, take specimen after cleansing meatus, or by catheter, and in staining decolorize with absolute alcohol after the acid (see pp. 163 and 285. In pus, faeces, and tissues : dissolve in antiformin as above, and examine deposit. In milk : centrifuge, take sediment and fat, and mix, make films, fix, remove fat with ether (not absolutely necessary), stain, and examine. Also inject o-i c.c, 1 c.c. and 3 c.c. of mixed sediment and fat into three guinea-pigs respectively ; kill in three weeks and examine peritoneum, post-sternal glands, pancreas, spleen, and liver. 2. Inoculation. — Select a guinea-pig and inject, sub- cut aneously or intraperitoneally, the fluid to be tested, or the washed sediment from an antiformin solution of tissues, faeces, or sputum. The animal usually dies in six weeks, if tubercle bacilli are in the injected substance, with local and glandular changes, the spleen showing numerous tuberculous nodules and being swollen as a whole. 3. Cultivation — Is made from the inoculated animal, or from antiformin solution sediments, or from sputum treated with 2 per cent ericolin. 4. Tuberculin Reactions. OTHER ACID-FAST BACILLI. Besides the bacilli of human, bovine, avian, and fish tuberculosis, there are other bacteria which are acid- fast, as already enumerated on page 163. Such are : Moeller's Timothy - grass bacillus I (from infusions of Timothy grass) , Moeller's Timothy-grass bacillus II (from the dust of a hay-loft), butter bacilli (isolated from butter by Petri, Rabinowitch, Korn, Tobler, Coggi, and others), mistbacillus (from dung by Moeller) . All these are mor- phologically very similar to the tubercle bacilli, are ex- tremely acid-fast, and produce lesions in guinea-pigs on injection, which closely resemble tubercles. They are, however, easily distinguished by their rapid growth on ordinary media, colonies being visible in twenty-four hours at 370 C. (in the case of the tubercle bacillus, the earliest is eight days), and by their growth in most instances at NON-SPORING BACILLI 285 room temperature. The cultures themselves are, however, similar to those of tubercle bacilli, and of one another. Johne's Bacillus is the bacillus of " chronic bovine pseudo-tuberculous enteritis," a disease characterized by corrugated thickenings of the mucous membrane of the small intestine (especially), the bacilli occurring in large numbers in the lesions and in scrapings from the surface. The bacilli are like the tubercle bacilli, but slightly shorter ; they are equally acid-fast. They have not yet been cultivated on artificial media. Bacillus Smegmatis (the smegma bacillus) occurs in large numbers in the preputial secretions of the male, the external genitals of the female, and within the folds of the thighs and buttocks. The bacilli are usually found in clumps on the mucous membrane, and occasionally in the superficial layers of the epithelium, both inside and outside the cells. They were first described by Lustgarten in 1884, who found them in a number of syphilitic lesions, and who thereupon believed them to be the cause of that disease. Further work by Alvarez and Tavel, Klemperer, and others, showed that they were harmless saprophytes. They are very similar to the tubercle bacillus, but are more varied in size (usually distinctly shorter), at times slightly curved and short. They are not easily stained, and once stained resist decolorization by acids, but not so strongly as tubercle bacilli. They are said to give up the stain to absolute alcohol, but contradictory statements, are made. On this basis is founded Pappenheim's method of staining, where a film stained with hot carbol-fuchsin is treated with absolute alcohol containing 1 per cent rosolic acid (corallin), methylene-blue to saturation, and 16 per cent of glycerin. The tubercle bacilli are red, the bacilli smegmatis blue. They are cultivated with great difficulty, and first on serum or ascitic media. They are non-pathogenic, so far as tested. Their growth on media is slow (five to six days) ; the colonies are yellowish- white, and corrugated like tubercle bacillus colonies. Bacilli of the smegma group have occasionally been demonstrated in sputum and in secretions from the throat and tonsillar crypts. Bacillus Leprae. — A bacillus closely resembling the 286 PUBLIC HEALTH BACTERIOLOGY tubercle bacillus in size and in acid-fastness. These bacilli usually stain uniformly, not showing beading. They are non-motile, non-flagellar, and non-sporing. They have not been successfully cultivated, and attempts to inoculate animals have failed. They are readily stained by Gram's method. They are found in large numbers in the cutaneous lesions of tubercular leprosy, and occur for the most part within the protoplasm of the round granulation tissue cells. They are also found in the lymphatic glands, and in smaller numbers in the liver and spleen. The spread of the disease is by the lymphatics. The earliest lesion is usually a nasal ulcer at the junction of the bony and cartilaginous septum. In the anaesthetic form, or nerve leprosy, the bacilli are found in the diffuse infiltrations in the nerves, rarely in the trophic lesions resulting. Lepers react to tuberculin, and 50 per cent are said to give the Wassermann reaction. The nasal mucus and saliva (in a less degree) are the vehicles by which the disease is spread. ^Diagnosis. — Animal inoculation is negative. ACTINOMYCOSIS, OR^THE^RAY FUNGUS DISEASE. Actinomycosis is a disease of cattle and man. [It occasionally affects sheep, dogs, cats, and horses. Its usual sites are the regions of the face, mouth, and pharynx. In cattle, the lower jaw is most frequently affected, the disease taking the form of tumour formation, the so-called " lumpy jaw." The tumours are often nodulated and consist of fibrous tissue with irregular abscess cavities throughout. When an abscess discharges, the pus is of a yellowish-green colour, slimy in character, and contains small granular bodies, visible to the eye and distinctly palpable, and of a pale sulphur colour. These granules are found on examination to be composed of rosette-like masses of the fungus actinomyces, or ray fungus, first described by Boellinger in 1877. I* *s now classed among the trichomycetes or higher bacteria, and by some as a true mould ; that is, forms composed of threads which show true branching and multiply by spore-shaped bodies, which usually appear in chains — the gonidia or spores. (Madura foot, or mycetoma, is similar in nature to actino- mycosis.) NON-SPORING BACILLI 287 Description. — An anaerobe of slow growth, growing best at 370 C. ; and in a shake culture in glucose agar, the colonies are most numerous 5 to 10 mm. below the surface of the medium, the inference being that a trace of oxygen is an advantage. The colonies are round, dense, and greyish-white in colour (chalky) ; sometimes they are rosette-shaped. Another variety has been described which is an aerobe, growing in three to four days to little transparent drops, becoming later amber, and then reddish- yellow in colour. This variety has been grown on gelatin, which it liquefies. In cultures, club-shaped forms have not been found in the aerobic variety, but have been noted by J. H. Wright in the anaerobic variety when grown in the presence of serum or other animal fluids. It is believed that several kinds have been described under the one name, and that further research is needed to differentiate these. The anaerobic form grows in broth, forming heavy flocculent masses (solid white mulberry granules) at the bottom of the tube ; no clouding nor surface growth. The fungus is described as having three forms : (1) Filaments, more or less radially arranged, 0-5 micron thick, and closely interlaced. These form the central core of the colony. (2) At the periphery, refringent club-shaped bodies, structureless and homogeneous ; whereas the filaments show a sheath, enclosing a granular protoplasm. (3) Spores or gonidia are coccus-like bodies, found between the filaments of the central mass ; are variously regarded as real gonidia, or as degeneration products, or con- taminating cocci. In cultures, gonidia are developed at the ends of the filaments, and such gonidia have a higher resistance to heat than the simple filaments, half an hour at 750 C. being required to kill spore-bearing cultures, and the same time at 650 C. for spore-free cultures. The filaments are Gram-positive and acid-fast. Pathogenicity. — In man, the disease tends to generalize ; in the ox, to remain local. The point of entrance in man is usually by a carious tooth, by the tonsil, or by some abrasion. The corresponding glands are next affected, and later metastatic abscesses are formed in the skin and elsewhere. The symptoms resemble those of chronic tuberculosis, for which the patient is usually treated. The 288 PUBLIC HEALTH BACTERIOLOGY disease is acquired probably from hay, straw, and grain r and possibly by milk of infected cattle. An actinomyces has been isolated from hay and straw, and in cattle, grains have been found embedded in the centre of growths. Inoculation of the ox has produced the disease ; in the smaller animals, characteristic colonies and lesions may follow, but little growth. Isolation. — May be easy or very difficult. The pus is washed in salt solution and sown in melted glucose agar. If much contamination is found, keep washed granules for several weeks in a dry state, and try again. Summary of the Final Report of the British Royal Commission on Tuberculosis Issued in June, 1911. The British Royal Commission appointed in 1901 to inquire into the relations of human and animal tuber- culosis, issued its final report in June, 191 1. The Commission was appointed on account of the diversity of opinion which was manifested at the International Congress on Tuberculosis, held in London in 1901, when the statement was made by Koch that human tuberculosis cannot be transmitted to cattle, and that bovine tubercu- losis is not dangerous to man. The results of the work of the Commission, and of much other parallel work, are to traverse directly both statements. The Final Report, extends to about fifty pages (there are 7 volumes of an Appendix), and may be usefully summarized thus : — The report is unanimous. It is based on the isolation of the bacilli from the lesions of the natural disease ; the investigation of the cultural characters of the bacilli isolated, and the study of their effects when introduced in varying doses and by several methods into different animals. The species of animals used have been cattle, rabbits, guinea-pigs, pigs, goats, chimpanzees, monkeys, horses, rats, mice, dogs, cats, and birds. The experimental methods of infection used were : subcutaneous, intravenous, intraperitoneal, and by feeding (oral). Inhalation was not tried. The findings are based on the researches of their own staff. NON-SPORING BACILLI 289 Three types of tubercle bacilli are described : — (i) Bovine tubercle bacilli — the only kind found in natural tuberculosis of cattle. (2) Human tubercle bacilli — the kind most commonly found in man, but not the only kind so found. (3) Avian tubercle bacilli — the only kind found in natural tuberculosis of birds. 1. Bovine tubercle bacillus is taken as the standard for comparison. Summary of its Characters.— (a). Cultural. — Grows slowly on serum, and at the end of two to three weeks shows on surface as a thin, greyish, uniform growth, not wrinkled nor pigmented. According to the rate and luxuriance of growth on glycerin media, bovine tubercle bacilli may be divided into three grades. Nevertheless it is insisted that rate and kind of growth should not be the sole basis of identification as bovine type, but only when considered along with results of inoculation experiments. (b). Effects on animals. — Produces characteristic effects when inoculated into calves and rabbits in certain doses. Calves : subcutaneous injection in neck of 50 mgr. of culture under three weeks old. causes severe general tuberculosis, starting at point of inoculation; and death usually within eight weeks. Rabbits : intravenous injection of o-oi mgr. or o-i mgr. of culture causes generalized miliary tuberculosis, ending in death within 5 weeks ; intraperitoneal injection of o*i mgr. — death in 13 to 48 days; intraperitoneal injection of i-o mgr. — death in 10 to 38 days; subcutaneous injection of 1 mgr. — death in 29 to 165 days; subcutaneous injection of 10 mgr. — death in 28 to 101 days. These results are very striking and definite, and along with cultural tests afford a trustworthy means of .recog- nizing bovine tubercle bacilli. Later it was considered sufficient to inoculate rabbits, as results are very reliable. (c). Other properties. — Subcutaneous inoculation in very small doses invariably produces acute tuberculosis in the chimpanzee, monkey, and guinea-pig. In the goat, the pig, and the cat, general tuberculosis is readily induced. The rat and the mouse are highly resistant to sub- 19 290 PUBLIC HEALTH BACTERIOLOGY cutaneous inoculation ; intraperitoneally, the bacilli mul- tiply in the body but do not produce tubercles. Dogs are highly resistant to subcutaneous inoculation, but succumb to general tuberculosis when large doses are given intravenously or intraperitoneally. In the fowl, intravenous injection of bovine tubercle bacilli caused death in 50 per cent, with wasting, cedema of lung, and pallor of liver. In a few, definite tubercles were found in the lungs and minute necrotic areas in the liver. Death is apparently due to toxaemia, as dead bacilli have the same effects. Intraperitoneally and intramuscu- larly, even in large doses, only local lesions are produced, and there is no dissemination. Horses : subcutaneously or orally, no progressive tuberculosis is produced. Intravenously, 10 mgr. cause death from acute tuberculosis in twenty days. (d). Stability in culture. — Subcultured for long periods, (one case, 1487 days = 4 years), no great loss of virulence was found. 2. Human tubercle bacillus. — The human type is taken as that bacillus which has been found in the majority of cases of human tuberculosis. Its chief characters are : on serum, it grows more rapidly than the bovine type, hence it is called " eugonic," as opposed to " dysgonic," the term applied to the bovine bacillus. On glycerin media, the growth tends to become wrinkled ; and on all media becomes pigmented to a greater or less extent. Its effects on animals place it in still greater contrast to the bovine type. Calves : subcutaneous injection in neck of 50 mgr. of culture under three weeks old, does not produce pro- gressive tuberculosis, nor does it kill. Only a local lesion results, which later becomes fibrous. In about half the cases, the infection did not extend beyond the nearest glands. Rabbits : intravenous injection of o-i mgr. to 1 mgr. of culture causes slowly progressive tuberculosis with limited lesions, and death (for the majority) after three months (thirteen weeks). Intraperitoneally, 1 mgr. ; animal alive after three months. Subcutaneous injection of 1 to 100 mgr. ; animals survived or were killed in 94 to 725 days. In certain cases 1 mgr. or o-i mgr. intra- NON-SPORING BACILLI 291 venously acted like the bovine type, killing with acute and rapid tuberculosis ; in these cases, however, o-oi mgr. never killed within three months, thus easily distinguishing the bacilli from those taken as the standard. Hence this dose is the best to use intravenously. Effects on chimpanzee and monkey in producing acute tuberculosis, are similar to those produced by like doses of the bovine tubercle bacillus. In guinea-pigs, it produces acute tuberculosis, but dura- tion of life is longer than with the same dose of bovine tubercle bacilli. In the goat, pig, and cat, great resistance is found, only slight retrogressive lesions being produced. In the dog, the effects are similar to the bovine tubercle bacillus, that is, there is great resistance to subcutaneous injection, but large doses given intravenously or intra- peritoneally cause generalized tuberculosis and death. In the fowl, the effects are the same as those produced by the bovine tubercle bacillus. In a horse, subcutaneous injection of 50 mgr. produced only local disease. The human tubercle bacillus has not shown any alteration in cultural characters on prolonged cultivation. Resume. — The human tubercle bacillus is distinguished from the bovine tubercle bacillus by (1) Its more ready growth on artificial media ; and (2) The results of inocula- tion into rabbits, calves, cats, pigs, and goats. They are alike in that they readily produce tuberculosis in chimpanzees, monkeys, and guinea-pigs, and in that the lesions produced in these animals are the same in distribution and structure. 3. Avian tubercle bacillus. — The avian tubercle bacillus forms a slimy, whitish growth, easily emulsified (difference from human and bovine). It grows badly on serum, but especially well on glycerinated media. Inoculation into animals produces effects markedly contrasting with those given by bovine and human tubercle bacilli. Fowls are very susceptible to intravenous, intramuscular, and subcutaneous inoculation of the avian tubercle bacilli, and also to feeding. In the former modes, the lesions are in the spleen and liver, and frequently in the lungs, cervical 292 PUBLIC HEALTH BACTERIOLOGY glands, muscles, and bones. After the feeding method, there are similar lesions, with, in addition, characteristic tuberculous lesions in the mucous membrane of the intestines. Parrots show similar results, but by feeding method do not so regularly show intestinal lesions. Parrots are susceptible to both the bovine and human tubercle bacilli, by inoculation and feeding ; the effects are similar to those produced by the avian type, except that the bovine type is apparently the most virulent for parrots. The rabbit and the mouse are the only two mammals in which the avian tubercle bacillus causes progressive tuberculosis. Rabbits : moderately large doses, by inoculation, produce a fatal issue ; the bacillus is less virulent than bovine tubercle bacillus, but more virulent than human tubercle bacillus. The distribution of the lesions differs most markedly from that set up by the bovine and human types. Intravenously, I to 10 mgr. lead to speedy death, with great multiplication of the bacilli in the organs, which show general pallor ; slight oedema of the lungs, slight enlargement (with tubercles) of the spleen. If the animal lives four to five weeks, the spleen is greatly enlarged owing to formation of tubercles, and tubercles are found in the liver and to a less extent in the lungs. In o-ooi mgr. dose, the disease is very chronic, and resembles that produced by subcutaneous injection, the joints being affected. Sub- cutaneous injection of doses of 50 mgr. down to a fraction of 1 mgr. causes very chronic disease, and the lesions have the same distribution, irrespective of size of dose : local lesion and nearest lymphatic glands ; liver and spleen rarely affected ; kidneys vary ; but most commonly and characteristically there is a tuberculosis of the joints of the limbs, which runs a chronic course. Joint tuberculosis occasionally follows intravenous injection of human tubercle bacillus, but has not been observed after sub- cutaneous injection of the rabbit with the human virus. It has however been noted after subcutaneous injection of the bovine type, when the animal survives for a long period, i.e., the disease is chronic. By feeding, similar lesions are produced, with local intestinal ones. NON-SPORING BACILLI 293 Mouse : General tuberculosis by subcutaneous or intraperitoneal injection, and by feeding. Calf, pig, goat, monkey, guinea-pig, horse, cat, and rat : all behave alike to the avian type. It never produces a progressive tuberculosis, but may kill them in a large dose given intravenously. Dogs : are immune intravenously. Chimpanzee : injected subcutaneously with 50 mgr. showed no lesion on death three years after. Vitality in Culture. — The bacillus was found alive in cul- ture after 1067 days (nearly 3 years). Chemical Properties. — The investigators were unable to detect any definite and constant bio-chemical character by which tubercle bacilli of one type can be differen- tiated from those of nother. BOVINE TUBERCULOSIS. — No further special investi- gation has been taken beyond that recorded in the second Interim Report, where in 30 cases of the natural disease in bovines, only one form or type of Bacillus tuberculosis was found. HUMAN TUBERCULOSIS. — In all, 128 cases of all forms of tuberculosis in man were investigated. Twenty of these were of lupus, and are treated separately because the recognition of the type of bacillus was surrounded with special difficulties. The 108 cases remaining are tabulated in full in the Report, and briefly in Table I., page 294. Of the cases of primary abdominal tuberculosis, the ages at death were as shown in Table II., p. 294. Twenty-four of the deaths were from some form of tuberculosis, the others from non-tuberculous affections. Of 15 of these cases, in which a plurality of lesions were examined on exactly parallel lines, 9 yielded none but human tubercle bacilli. In 12 cases in which a single lesion in each instance was examined (mesenteric glands in II, cervical gland in 1), 4 yielded human and 8 bovine tubercle bacilli. Two cases yielded mixed human and bovine types, in one from the mesenteric glands alone, and in the other, also from the retroperitoneal glands. The cervical gland cases show 3 bovine infections out of 294 PUBLIC HEALTH BACTERIOLOGY Table I.- — -Cases of Human Tuberculosis other than Lupus Type of Bacillus Nature of Cases Cases found Bovine Human Mixed i. Primary pulmonary tuberculosis: (phthisis pulmonalis) A . Tissues examined post mortem r(lung in 13 and bronchial gland alone in 1) 14 — M — B. Sputum from other cases 28 2 26 — 2. General tuberculosis : various tissues 3 3 — . 3. Tuberculous meningitis : cerebro- spinal fluid in 2 3 ■ 3 — 4. Bronchial gland tuberculosis 5 3 2 5. Cervical gland tuberculosis : (removed by operation) 9 3 6 6. Primary abdominal tuberculosis : mesenteric glands and other tissues 29 M 13 2 7. Joint and bone tuberculosis : scrapings and abscesses 14 — 13 I 8. Tuberculosis of testicle (1), kidney (1), and suprarenal (1) . . 3 — ■ 3 Totals 108 19 84 5 Notes. — The cases in group 1 A were all clinically cases of con- sumption, in which death resulted from the pulmonary disease. The two patients who had bovine tubercle bacilli in their sputum died subsequently, but no post-mortem examination was obtained. The ages in the sputum cases were : 16 to 25 years, 19 cases ; 26 to 33 years, 8 cases ; and 50 years, 1 case. Table II. — Primary Abdominal Tuberculosis. Type of Virus found PRESENT Bovine Human Mixed 1 to 3 years 3 to 5 „ ■ 4 to 5 „ . 7 » • 8 „ . 15 „ • 18 „ • 70 „ • IO 3 I 8 3 1 1 I I 18 3 3 Tc >tals M 13 2 29 NON-SPORING BACILLI 295 9 investigated, and these too are ascribed to pharyngeal or buccal infection, that is, alimentary infection. With the primary abdominal cases, this gives 38 cases of tuberculosis of presumably alimentary infection, in which the bovine bacillus alone was found in 17 instances, the human in 19, and a mixed infection in 2. Summary. Cases Exam- ined. Type of Virus Found. Bovine. Human. Mixed. Avenue of infection : (a) Respiratory tract (presum- ably) (b) Alimentary tract (presum- ably) 47 38 2 *7 43 19 2 2 Age : (a) Adolescents and adults (6) Children 55 53 3 16 50 34 2 3 Lupus. — Out of 20 cases investigated, the virus was decided to be bovine in 9 cases, and to be the human type in 11. All but three of the cases presented difficulty in the detection of the type of bacilli. One was undoubtedly bovine by the already decided-on tests, two were likewise human ; but the remaining 17 furnished bacilli which conformed to the one type in some particulars and to the other in other particulars. Thus the other 8 finally called bovine showed the cultural characters of that type, but a lowered virulence for the calf and also for the rabbit, monkey, and guinea-pig. Two had their virulence raised by passage through the calf and rabbit, bringing it up to that of the bovine type. Those ascribed to the human type (11), but not typically so, had lowered virulence for all the test animals or some of them. The general result therefore seems to point to the existence of bacilli outside the types chosen ; these may be new types, or members of the other types with degraded virulence. Tuberculosis in Swine. — In 59 cases investigated of 296 PUBLIC HEALTH BACTERIOLOGY tuberculosis of all kinds in swine, the bovine virus was found in 50 cases, the human in 3, the avian in 5, and a mixed avian and bovine virus in 1 case. In 33 of these cases the tuberculosis was generalized, and in 32 of these the infection was bovine, the other being the one of mixed bovine and avian infection. The other 26 cases showed local tuberculosis. Conclusions : All three types of bacilli are capable of infecting the pig, but the bovine bacillus is the one much most frequently found, and in many instances it produces a severe and generalized disease. Tuberculosis in Horses. — In most cases of tuberculosis in horses it is primarily an affection of the glands and organs in connection with the alimentary tract ; the abdominal organs chiefly. This was so in the five cases investigated. In all, bacilli were recovered which were of the bovine type culturally ; and three of them also had the bovine virulence. The other two had diminished virulence for the test animals in the test doses ; passage experiments raised this to that of the bovine virus. Tuberculosis in other Animals. — In a gnu which died from generalized tuberculosis in the London Zoological Gardens, the human virus only was found. In an antelope, killed when ill, in the same Gardens, where it had been many years in captivity, extensive tuberculosis, with cavities in the lungs, was found. The human virus alone was isolated. In a rhesus monkey, killed when ill at the quarantine station at Isleworth, tuberculosis of the lungs was found, and some elsewhere. Here too only the human virus could be got. A chimpanzee died of acute miliary tuberculosis at the quarantine station. The tuberculosis started from the alimentary tract, and from a mesenteric gland the human virus was obtained. A cat, suffering from naturally acquired tuberculosis, was investigated. A mesenteric gland gave a bacillus of the bovine type in growth and virulence. Tuberculosis in Birds. — In 9 cases investigated (3 fowls, 3 pheasants, 1 pigeon, 1 demoiselle crane, 1 Senegal touracou) of tuberculosis occurring naturally in NON-SPORING BACILLI 297 birds, in every one the virus found was of the avian type. In tuberculosis in pigs, the avian virus was found on 6 occasions in the submaxillary lymphatic glands ; in 5 alone, and in I case in association with the bovine virus. (Query. — Is this due to special exposure of the pig to the farmyard dust, and to its high body temperature ?) Behaviour of the Bacilli and their Fate in the Tissues of Inoculated Animals. — From a number of experiments detailed, the Reporters conclude that after subcutaneous inoculation of human and bovine viruses, rapid and abundant distribution of bacilli over the body takes place, provided the dose is large and the tissue condi- tions of the animal such as to allow the inoculation to take full effect. This is essentially a mechanical dispersion by the blood and lymph channels of a considerable proportion of injected bacilli, and occurs speedily after their insertion. The resulting acute tuberculosis is at first in strong contrast to the similar but more slowly developing generalized disease induced by smaller experimental doses, or such as occurs most commonly in nature. This more slowly developing form is due to a dispersion of bacilli, but not necessarily those primarily invading the animal ; more pro- bably their progeny, which have been able to get through the barriers and into the blood-stream. Feeding Experiments. — In two pigs fed with large doses of bovine virus, the bacilli were demonstrated (by inoculation of guinea-pigs) in the submaxillary and mes- enteric glands and lungs in seven and thirteen days after ingestion, but not in the other organs. In seven pigs fed with human virus, in from two to twelve days after ingestion the same distribution was proved in two ; in the other five the virus was found in the glands alone. In a goat, eight days after ingestion of human bacilli, they were found in the submaxillary glands, mesenteric glands, and lung. In a cat fed with the same bacilli, in nine days they were found in the submaxillary and mesenteric glands, lung, liver, and spleen. Three rhesus monkeys, each fed with 50 mgr. of human tubercle bacilli, were killed ; 1 in two days, showed no bacilli in the glands or internal organs; 1 in four days, showed bacilli in the mesenteric glands, liver, spleen, and lungs 298 PUBLIC HEALTH BACTERIOLOGY and the third in six days, showed bacilli in the sub- maxillary and mesenteric glands and spleen, the lung condition, owing to the premature death of the guinea- pigs used for the test, not being determined. Excretion of tubercle bacilli in milk was tested by injection of cultures into healthy cows and goats. Subcutaneous injection of ioo mgr. of bovine culture into a healthy milch cow caused its death thirty days later of general tuberculosis. The udder was normal both to the naked eye and on microscopical examination, yet guinea- pigs fed with the animal's milk, by the end of the first week after injection and subsequently, developed tuberculosis. Two other cows were injected with human tubercle bacilli. One received ioo mgr. subcutaneously ; tubercle bacilli were recovered from her milk in twenty-four hours, and the milk in small doses caused tuberculosis in guinea-pigs at every time of testing right up to 155 days later, when the cow was killed and the udder was found normal. The other received 10 mgr. intravenously, and the milk contained the bacilli in twenty-four hours and up to fourteen days later, but not subsequently. The animal was killed in 182 days, and showed no tuberculous lesions. Six milch goats were similarly tested with bovine and human bacilli, with confirmatory results. In 12 experiments on heifers, subcutaneous injection of human virus (2), lupus virus (8), and bovine virus (2), in 50 to 100 mgr. doses, the heifers were killed in 62 to 127 days afterwards, and in eight cases tubercle bacilli were found in the sinuses of the undeveloped udder of the animals ; in four, in such numbers as to suggest multipli- cation in these sinuses. Modification of Bacilli. — Tests by cultural processes and by long-continued passage through animals have failed to effect any change of type from bovine to human, bovine to avian, human to avian, and vice versa. Certain difficulties were encountered with bacilli in lupus and in the horse, but while the Reporters think that further investigations may possibly disclose additional variations in the types of bacilli, they do not as a result of their investigations feel disposed to add a plurality of new types to the three already described by them. They are NON-SPORING BACILLI 299 not prepared to deny that the transmutation of one type into another may occur in nature, in view of the instances in which one and the same human body yielded both types. Replies to the Terms of Reference. — The questions referred to them for investigation and report were as follows : — (i) " Whether the disease in animals and in man is one and the same. (2) Whether animals and man can be recipro- cally infected with it. (3)- Under what conditions, if at all, the transmission of the disease from animals to man takes place, and what are the circumstances favourable or unfavourable to such transmission." 1. Morphologically, as grown on serum, the human and bovine types described are indistinguishable, but they are appreciably different in respect of their cultural characters and their capacity for causing disease in various species of animals. The question of the identity or non-identity of these two types clearly depends therefore upon the importance which it is permissible to attach to their cultural and pathogenic differences, and this depends on the fixity or variability of the differences in question. Though in the investigations no case was observed in which the mode of growth of one type was so modified that it was indistinguishable from the mode of growth of the other type, yet the bacilli referred to as the bovine type show so much variety among themselves as to luxuriance of growth, that the gap which separates those of that group which grow most luxuriantly from the human type, is not a wide one. Again, as regards pathogenicity, it is more a matter of degree than of difference. The bovine tubercle bacillus produces a fatal tuberculosis in cattle, rabbits, guinea- pigs, chimpanzees, monkeys, goats, and pigs. The human tubercle bacillus readily produces a fatal tuberculosis in guinea-pigs, chimpanzees, and monkeys, and in large closes, only slight and non-progressive lesions in cattle, goats, and pigs. Its effects on rabbits are not uniform, for while in the majority of cases these animals are only slightly affected, in some cases extensive and fatal tuberculosis results. 300 PUBLIC HEALTH BACTERIOLOGY In other words, guinea-pigs, chimpanzees, and monkeys are all highly susceptible to the effects of human or bovine tubercle bacillus ; and the diseases produced in these animals by both types are histologically and anatomically identical. In man, experiment is not permissible, and no oppor- tunity has offered of generalized disease set up by accidental infection with the bovine tubercle bacillus. Nevertheless many cases of fatal tuberculosis caused by the bovine tubercle bacillus and nothing else have been investigated. Compared with parallel cases caused by the human tubercle bacillus, the two groups of cases were alike in their clinical histories and their fatal termination, and were indis- tinguishable anatomically when the lesions were examined after death. Man must therefore be added to the list of animals notably susceptible to bovine tubercle bacilli. Are these two types, then, varieties of the same organism ? This is the conclusion, in spite of the failure to transmute the one into the other. And, as a corollary, the lesions they produce, whether in man or in other mammals, are manifestations of the same disease. Whatever difference of opinion may be held on this conclusion, in a considerable proportion of cases of human tuberculosis the disease is one and the same as bovine tuberculosis, being caused by bacilli which are in every respect indistinguishable from the bacilli which are the cause of tuberculosis in cattle. In all such cases therefore the disease must unquestionably be pronounced as one and the same. As regards avian tuberculosis, there does not appear to be sufficient evidence at present to answer the question in the affirmative. 2. The conclusion reached is that, excluding the fowl and other birds, mammals and man can be reciprocally infected with tuberculosis. The transmission to man has been conclusively shown by the study of fatal cases of tuberculosis, mostly in children ; and from man to mammals, by feeding experiments. 3. Conclusions : — (i.) Unmodified avian tubercle bacillus is a negligible factor in the production of human tuberculosis. (ii.) It cannot be affirmed with confidence that man is NON-SPORING BACILLI 301 wholly free from risk of infection, through animal food, with that type of bacillus to which he is most prone, namely the human type ; though the degree of danger to him in this sense must for the present remain undetermined. The pig, though experimentally it fostered human bacilli in a minor degree only, may have to be regarded as a possible source of this kind of infection, since particular glands of the pig's body, in which human tubercle bacilli have occasionally been found, are likely to enter into certain prepared foods. (iii.) (a) The pig is a potential source of infection of man with bovine tubercle bacilli. This bacillus was present in 50 -f- 1 cases of tuberculosis in pigs, out of 59 cases of tuberculosis investigated. In 32 + 1 of these cases, it caused generalized tuberculosis, and in 18 cases, local tuberculosis. (The -f 1 case was one of mixed bovine and avian infection.) There is no reason to suppose that the bovine tubercle bacilli are rendered less infective to human beings by previous residence in the tissues of the pig. (b) The actual number of cases representing the various clinical manifestations of tuberculosis commonly found in man, on which the conclusions are based, is. 128. So far as these have been examples of tuberculosis in adults, and especially when they have been cases of pulmonary tuberculosis, the lesions of the disease, when fatal, have been referable to the human tubercle bacillus, with but few exceptions. In human abdominal tuberculosis, the experience has been very different, especially as regards children. Of young children, dying of primary abdominal tuberculosis, the fatal lesions could be referred to the bovine tubercle bacillus, and it alone, in nearly one-half of the cases. In cervical-gland tuberculosis, in children, and often also in adolescents, a large proportion of the cases examined could be referred to the bovine tubercle bacillus. In lupus, too, in the cases examined occurring in adolescents and children, the amount of infection with the bovine type was marked. Whatever therefore may be the animal source of infection with the bovine type of bacillus in adult and adolescent mankind, there can be no doubt that a consider- 302 PUBLIC HEALTH BACTERIOLOGY able proportion of the tuberculosis affecting children is of bovine origin, more particularly that which affects primarily the abdominal organs and the cervical glands, and further that primary abdominal tuberculosis, as well as tuberculosis of the cervical glands, is commonly due to ingestion of tuberculous infective material. In what way are children most likely to obtain a large and fatally infective dose of tubercle bacilli ? To this question there can be but one answer, namely, the evidence accumulated goes to demonstrate that a considerable amount of tuberculosis in children is to be ascribed to infection with bacilli of the bovine type, transmitted in meals consisting largely of the milk of the cow. The child may be subjected to this feeding with infective material, and not develop a fatal tuberculosis ; but still be injured, although it recover. Many cases of abdominal tuberculosis in children recover, and the proportion of bovine to human bacilli in these has not been estimated ; and of cervical-gland tuberculosis, nearly all make some kind of recovery, with varying degrees of disfigurement ; and a similar statement may be applied to lupus. In adult and adolescent mankind (excluding lupus, in which, out of 10 cases, three yielded bacilli culturally bovine but with less virulence for the calf and rabbit than the bovine tubercle bacillus), fatal lesions due to bovine bacilli have been found, rarely in adolescents, and extremely rarely in adults. Yet, although of 55 cases scrutinized of tuberculosis in adults and adolescents, only 5 yielded bovine bacilli, it cannot be said that this figure adequately represents the proportion of like cases among the tubercu- lous population generally. In view of the evidence adduced, the following pronounce- ments on administrative measures required at present to obtain security against transmission of bovine tubercle bacilli by means of food, are called for : — In the interests of infants and children, and for the reasonable safeguarding of the public health generally, it is urged that existing regulations and supervision of milk production and meat preparation be not relaxed. On the contrary, Government should cause to be enforced NON-SPORING BACILLI 303 throughout the kingdom food regulations, planned to afford better security against the infection of human beings through the medium of articles of diet derived from tuberculous animals. More particularly it is urged that action in this sense should be taken, in order to avert or minimize the present danger arising from the consumption of infected milk. Certain facts observed in reference to the elimination of bovine tubercle bacilli by the cow in her milk, are of such importance that they formed the subject of the third Interim Report, and deserve repetition here. i. Bovine tubercle bacilli are apt to be abundantly present in milk, as sold to the public, when there is tuberculous disease of the udder of the cow from which it has been obtained. This fact is generally recognized, though not adequately guarded against. 2. Bovine tubercle bacilli may also be present in the milk of tuberculous cows presenting no evidence whatever of disease of the udder, even when examined post mortem. 3. In tuberculous cows, the milk leaving the udder may not contain tubercle bacilli, and yet it may and frequently does become infective by contamination with the faeces or uterine discharges of such diseased animal. Convinced that measures for securing the prevention of the ingestion of living bovine tubercle bacilli with milk would greatly reduce the number of cases of abdominal and cervical gland tuberculosis in children, the Reporters advise that such measures should include the exclusion from the food supply of the milk of the recognizably tuberculous cow, irrespective of the site of the disease, whether in the udder or in the internal organs. (A memorandum is appended in which reference is made to immunity experiments, on which no opinion is expressed, but which are fully reported in Vol. iii. of Appendix to this Report ; and to several other subsidiary experiments.) CHAPTER XIV. SPORING BACILLI. Sporing bacilli are comprised in two groups : — i. Aerobic (facultative anaerobes). Non-motile : anthrax, anthracoides and radicosus. Sluggishly motile : mycoides, ramosus, vulgatus, mesentericus. Actively motile : subtilis, megatherium. AH the above are Gram-positive ; gelatin-liquefying ; non-indol-forming, and non-gas-forming in glucose or lactose ; coagulate milk slowly with little acid and then digest the clot ; digest blood serum. 2. Anaerobic (strictly). — Subcutaneous injection into animals causes : — (i). No particular symptoms at site of inoculation, but absorption of the soluble toxin causing — (a) general symptoms of tetanus, B. tetani ; (b) botulism, pupillary symptoms, paralysis of tongue and pharynx, cardiac and respiratory failure, B. botulinus. (ii). Local symptoms marked at the site of inoculation, causing haemorrhagic emphysematous oedema ; (a) motile ; spores oval and central, B. cedematis maligni ; spores oval and excentric, B. anthracis symptomatici ; spore near one end, B. enteritidis sporogenes ; (b) non-motile; B. aerogenes capsulatus of Welch and Nuttall. SPORE-BEARING AEROBIC BACILLI. Bacillus Anthracis is the cause of anthrax, a disease primarily of the herbivora, cattle and sheep, but occurring also in horses, pigs, and goats. Man is susceptible, and contracts it either directly from the living or dead animal, or from hides, wool, horse-hair, or dust arising from these. It assumes two forms, external anthrax or malignant pustule, and internal anthrax which in man takes the form of wool-sorter's disease and the form of intestinal anthrax, in which the symptoms are more like those of acute poisoning. In human anthrax, bacterial invasion of the blood only SPORING BACILLI 305 occurs late in the disease ; in animals, on the contrary, the blood-invasion is early. The Algerian sheep and the white rat have a high degree of immunity. Pollender first described the anthrax bacillus as occurring in the blood of animals succumbing to splenic fever. Rayer and Davaine repeated the observation in 1850 (a year later) ; Brauell, in 1857, found the bacilli in the blood of a man affected with anthrax, and Davaine gave everything but absolute proof that they were the exciting cause of anthrax. Koch, by succeeding in getting a pure culture on the aqueous humour of an ox's eye, was able to prove its specificity. He also added largely to the knowledge of its life-history, and particularly to the mode of formation of spores. Description. — B. anthracis is a straight rod, non-motile, with square or concave ends, 4-5 to 10 micra long by 1 to 1-5 micron thick; forming chains in cultures, and sporing by oval spores one to each rod ; placed about the centre of the bacillus, and of about the same diameter, and highly retractile. Gram-positive ; gelatin-liquefying, and said at times to possess a capsule when recovered from tissues or blood, or grown on latter. Cultures. — Grows well on all media, best at 37-5° C, but also from 12 ° to 45 ° C. In broth: a heavy flocculent sediment, slight pellicle, remainder clear. In gelatin stab : an invreted fir-tree growth, with gradual fluidification. In gelatin plate : colonies develop within 24 to 48 hours as opaque white pin-head discs, later becoming larger and less regular, and under the microscope showing a hair-like tangle of threads — the so-called Medusa head. In agar plate : the colonies magnified thirty times show wavy wreaths like locks of hair, the whole colony being probably one long thread. Such colonies are very suit- able for making impression preparations, and in such the wreaths are seen to be made up of bundles of long filaments lying parallel with one another, each filament consisting of a chain of bacilli. On potato : a thick white felted mass, useful for studying porulation. 20 306 PUBLIC HEALTH BACTERIOLOGY Spores. — Only produced in the presence of oxygen (free), and hence not formed in blood of infected animals while in the unopened vessels or tissues. For this reason it is advised to cut into an animal dead of anthrax as little as possible, and to be specially careful not to spill the blood. The spores are very resistant, keeping for twenty years. They are killed by dry heat at 1400 C. (2480 F.) in 3 hours, and live steam at ioo° C. in 5 to 10 minutes, or boiling water for 1 J hours. Their behaviour to chemical disinfectants is variable, some strains resisting 1-20 carbolic acid for forty days, while others are destroyed by the same solution in two days. Corrosive sublimate, 1-2000, kills most strains in 40 minutes. Direct sunlight destroys anthrax spores within 6 to 12 hours. Creolin (10 per cent) kills anthrax bacilli in 10 to 20 minutes, but anthrax spores can survive in a 60 per cent solution of creohn. Freezing has little effect on their vitality. Spores are formed best at 300 C, and by keeping the bacilli at 420 C. for eight days, the power of sporulation is lost, and is only regained by passing the bacilli through a series of animals. Anthrax spores are often used for testing the value of " germicides." To do this, sterile silk threads are steeped in an emulsion of an anthrax culture and are dried over strong sulphuric acid in a desiccator. They are then placed in a solution of the " germicide " for a certain time, well washed with water, and laid on the surface of agar medium or dropped into broth, and incubated to see if any growth occurs. The culture used is first tested for spore formation. Pathogenicity. — For man : great. For animals : mainly for cattle and sheep. In the German Empire in 1899, the following cases were reported : 3678 cattle, 307 sheep, 282 horses, 61 swine, and 6 goats. In Great Britain, in the ten years 1896 to 1905, the total reported " outbreaks " in animals were 6203, and the number is increasing. In man, 512 cases were reported in 1901 to 1910, and of these 120 were fatal. Internal anthrax is usually fatal. In the external form, head and neck cases show a mortality of 85 per cent ; and hand and arm cases 12 per cent. SPORING BACILLI 307 Rabbits, guinea-pigs, and white mice are all very susceptible, the mice most so. Rats resistant, especially the white rat ; dogs more so. Birds are highly immune, also amphibians (but toads are said to be very susceptible). Toxins. — No toxins have yet been isolated, though it is highly probable that both extra- and intra-cellular toxins exist. Vaccination. — In France, a death-rate from anthrax of 10 per cent among sheep and 5 per cent among cattle compelled attention to the problem of providing protection. Pasteur, in 1881, introduced his method by the use of two vaccines : (1) A broth culture of bacilli, whose virulence was reduced by being incubated at 420 C. for twenty-four days, and so made non-fatal to guinea-pigs but still fatal to white mice — premier vaccin ; (2) A broth culture, incu- bated as above for twelve days, which would kill guinea- pigs but not rabbits — deuxieme vaccin. A sheep was inoculated in the subcutaneous tissues on the inner side of the thigh with 5 drops of the premier vaccin. Twelve days later a similar inoculation of the deuxieme vaccin was given, and fourteen days later still an injection of an ordinary virulent culture produced no ill result. The method has given excellent results, and the immunity lasts about a year. Passive Immunization. — Sclavo produced a serum from highly immunized asses, which has strongly protective and curative properties, and is used in the treatment of anthrax in man. In malignant pustule, four doses of 10 c.c. are injected into the abdominal wall, and if necessary repeated on the following day. Sclavo does not advise excision of the pustule. Sobernheim uses serum from sheep. Isolation of B. anthracis from hairs, etc. : Add 5 grm. to broth and shake. Incubate : not a pure culture. Heat to 8o° C. for 30 minutes ; all non-sporing organisms killed. Take twenty samples of 1 c.c. on agar and grow. Infect animals and see if pathogenic. Plate again on second day. Diagnosis. 1. In a case suspected to be malignant pustule, diagnose by (1) Making films from the fluid in the vesicles or from scrapings, and staining with watery methy- lene-blue, and also by Gram (be careful in scraping a pustule 308 PUBLIC HEALTH BACTERIOLOGY before excision, not to manipulate it roughly, or bacteria may enter the circulation) ; (2) Making cultures from similar material, by successive strokes on agar tubes or plates ; (3) Inoculation of the cultures into a guinea-pig or mouse, subcutaneously. If anthrax bacilli are present the animal usually dies within two days, and post mortem the tissues around the site of inoculation show intense inflammatory oedema, swelling, and gelatinous change, with small haemorrhages. On microscopic exami- nation, numerous bacilli are seen. The internal organs show congestion and cloudy swelling, and sometimes small haemorrhages, and their capillaries contain enor- mous numbers of bacilli, so that they appear as if injected with them. The spleen is notably enlarged (especially in the ox dead of anthrax, being two to three times its natural size, hence the name "splenic fever"), is of a dark-red colour, and on section is soft and friable, at times almost diffluent. Films from the pulp contain enormous numbers of bacilli mixed with red cells and leucocytes of the lymphocyte and large mononuclear varieties. The lymphatic system is generally much affected, the glands and vessels being swollen and containing bacilli in very great numbers. The intestines are enormously congested, the epithelium is more or less desquamated, and the lumen filled with a bloody fluid. (Muir and Ritchie.) 2. Methylene-blue Reaction. — Depends on the disintegra- tion of the capsules of the bacilli, which occurs when these are imperfectly fixed. It serves for the easy recognition of anthrax bacilli in blood and other bodily fluids, where putrefactive and other bacilli are present. Dry a loopful of blood on a slide ; hold it for one second in the flame ; repeat three times. Stain for a few seconds in old solution of methylene-blue, wash in water, and dry. Examine dry and without a cover-glass, when between and near the bacteria, violet or reddish-purple tinted granular or amorphous matter is seen. (M'Fadyean's test.) Capsules can be demonstrated in smear preparations from organs, by staining in 2 per cent watery solution of methylene- violet (heating). Wash in water for 2 seconds. Wash in 1 per cent acetic for 6 to 10 seconds. Wash in SPORING BACILLI 309 water and examine in water-drop. Another method is to stain (without fixing film) in a cold saturated solution of gentian-violet in formalin. Examine in water-drop. Prevention of Anthrax. — The Home Office Order No. 1293, dated Dec. 12th, 1905, on this subject, is made under Section 79 of the Factory and Workshop Act, 1901. It provides for the prevention of dust from wool or hair by ordering the opening and sorting to be done only (1) after steeping in water ; or (2) over an efficient opening screen, with mechanical exhaust draught, in a room set apart for the purpose and in which no other work than opening is carried on. Mohair, other than Van mohair, only needs to be sorted over a down draught. Van mohair, Persian locks, and Persian, must all be steeped before being opened. Alpaca, pelitan, East Indian cashmere, Russian camelhair, Pekin camelhair and Persian [or so-called Per- sian, if to be sorted or willowed] must be steeped before opening, or opened over efficient opening screen. It also provides for the use of overalls and respirators, cubic space per person (1000 c. ft.), temperature of room (not lower than 500 F.). Treatment of cuts and sores, washing of hands, burning of dust and other refuse, rules for treat- ment of damaged hair, proper kind of screen and board, lime-washing of rooms yearly, daily disinfection of floors and sweeping thereafter, etc., etc. Anthrax orders are issued by the Board of Agriculture under the Diseases of Animals Act, 1894, and provide for the precautions to be used in treatment of a sick animal, and (1) the burial at a depth of not less than 6 ft., with 1 ft. of lime beneath and above the carcase of an animal dead of anthrax, or (2) the destruction of a dead animal by heat or chemical agents. The local authority must carry out the provisions of the order in these and other particulars. Bacteria closely resembling B. Anthracis : — 1. B. Anthracoides. A Gram-positive bacillus; ends more rounded than those of B. anthracis ; growth more rapid ; gelatin liquefaction more rapid ; non-pathogenic. Otherwise indistinguishable from B. anthracis. 2. B. Radicosus. Cultivated from water-supplies. 310 PUBLIC HEALTH BACTERIOLOGY Larger, and more variation in size of individuals ; grows best at room temperature ; non-pathogenic. 3- B. Subtilis. The common bacillus of hay infusion, and found as a saprophyte in old wounds and infected sinuses ; gives a heavy tenacious pellicle in broth ; spores germinate equatorially ; gelatin and casein are liquefied more rapidly than by B. anthracis ; is actively motile in young cultures ; non-pathogenic. Pathogenicity for man is now alleged, it having been isolated from a case of panophthalmitis in pure culture. 4. B. Mycoides (earth bacillus). Is obtained from the surface of the earth of cultivated fields or gardens. Grows best at 180 C., and on gelatin shows a mould-like growth. About the size of B. anthracis, which it resembles greatly. Grows in threads ; motile ; non-pathogenic. 5- B. Megatherium. 10 micra x 2-5 micra. First found on boiled cabbage leaves. 6. B. Vulgatus (potato bacillus). Grows rapidly on potatoes, showing marked wrinkling. Small thick rods with rounded ends, in pairs or fours. SPORE-BEARING ANAEROBIC BACILLI. Methods of Anaerobic Culture. 1. Liquid media : Boil vigorously for 15 minutes, to drive out dissolved oxygen ; cool, and put layer of sterile oil on surface. (Pasteur.) 2. Piece of sterile mica on surface of agar or gelatin plates. (Koch.) 3. Deep inoculation in solid media, recently boiled for 15 minutes and cooled rapidly in ice, to prevent absorp- tion of oxygen. It is advantageous to have 1 per cent of glucose in medium. The tubes are inoculated by deep stabs, and the top of the medium is covered with a thin layer of agar, gelatin, or oil. and the tube capped with rubber or sealed with sealing-wax. (Liborius.) 4. The solid medium may be inoculated before it solidifies, — a " shake culture " — as in the original method of Liborius. The colonies which develop are fished out after breaking the tube. 5. Tube fitted with two-holed cork and two tubes. SPORING BACILLI 311 Pass in H or N until all the air is displaced, and then seal ends of tubes in flame. 6. Buchner's Tube. — A wide tube with a constriction near the closed end, so that an ordinary culture tube can be inserted and is held up by the constriction. In use, i grm. of solid pyrogallic acid and 20 c.c. of 10 per cent KOH are put into the bulb, the previously inoculated tube is inserted loosely plugged, and the Buchner tube is plugged and sealed with melted paraffin or closed with a rubber cap. The solution in bulb absorbs oxygen. The method can be used without the special tube, with a tall glass jar or a desiccator. It is better to add the alkali by pipette after the inoculated tube has been inserted. 7. Bulloch's Method. — A bell jar with an inlet and an exit tube, both having stopcocks. The bell jar is firmly bound down to a glass plate by ung. resinae. Before fixing, a glass dish is set on the plate, and 3 to 4 grm. of pyro- gallic acid are heaped to one side of it. Culture plates or tubes in a beaker are rested on a tripod stand placed in the dish. The bell jar is now fixed on so that the inlet tube will end in the dish at the side away from the pyrogallol. Hydrogen is passed in, and then a solution of KOH (109 grm. in 145 c.c). 8. Vacuum Method. — Desiccator with stopcock. Exhaust air by burning alcohol on soaked filter-paper put into jar. Stopcock is needed to release pressure when opening. B. Tetani. The cause of tetanus or lockjaw, a disease characterized by the gradual onset of general stiffness and spasms of the voluntary muscles, beginning in the jaw muscles and those of the back of the neck. The disease is usually associated with a wound received four to fourteen days previously and infected with earth or dung. The majority of cases are fatal. Kitasato first isolated the bacillus (in 1889) and in this fashion : — Pus from the local suppuration of mice inoculated from a human case, was smeared upon the surface of agar slants. These were permitted to develop at 370 C. for 24 to 48 hours. At the end of this time the cultures were subjected to a temperature of 8o° C. for 1 hour. This destroyed all non-sporulating bacteria as 312 PUBLIC HEALTH BACTERIOLOGY well as aerobic spore-bearers which had developed into the vegetative form. Agar plates were then inoculated from the slants and incubated in a hydrogen atmosphere, and on these tetanus bacilli grew. Rosenbach had previously pointfd out the terminal spore formation of B. tetani, and Nicolaier had described the bacillus, but could not grow it in pure culture. Description. — A slender bacillus, 2 to 5 micra long x 0-3 to 0-8 micron thick, with somewhat rounded ends. In young cultures it is slightly motile, and by special staining numerous peritrichal flagella are seen. In 24 to 48 hours the bacilli develop spores which are at full size three to four times the thickness of the bacillus in diameter, and are formed at one end of the bacillus. The bacillus and spore thus give the characteristic drumstick appearance. It is easily stained with the usual dyes, and is positive to Gram's method. Detached flagella often become massed together in the form of spirals, not unlike spirochaetes. In specimens stained with watery solutions of gentian-violet or methylene - blue, the bacillary protoplasm stains uniformly, but any spores are unstained except at the periphery, and so look like rings. With carbol-fuchsin and time, the spores become uniformly coloured. Spores may be found free from the bacilli in which they were formed. The bacillus liquefies gelatin slowly, also blood serum, but does not coagulate milk. It is a strict anaerobe (obligatory), but can be habituated to aerobic life, though with loss of pathogenicity and toxin-forming power. Grows moderately in aerobic conditions where other organisms which use up the oxygen supply are present (symbiosis). Its growth is aided in all media by slight alkalinity, presence of glucose, maltose, or sodium formate (1 to 2 per cent), which act as reducing agents. With carbohydrates it produces acid. In gelatin and agar, moderate amounts of gas are produced, chiefly CO 2 ; but other substances which are volatile and cause a characteristically unpleasant odour are formed. This is described as a peculiar burnt odour, and as that of putrefying organic matter, and is said to be largely due to H2S and CH3.SH (methyl mercaptan). Cultures.— In deep glucose gelatin stab : growth begins SPORING BACILLI 313 one inch or so below the surface, in fine straight threads, radiating from the needle track. Slow liquefaction, with slight gas formation, takes place. In agar stab (glucose agar) : the growth is somewhat similar: In glucose broth : slight clouding, with later a thin powdery deposit on the walls of the tube. Ordinary broth is preferred for toxin production. On blood serum : growth with liquefaction takes place. In milk : acid is formed but no clot. On potato : growth is delicate. On agar plates : colonies show a compact centre, with loose feathery outline not unlike B. subtilis or anthrax. Spores— Resist dry heat at 8o° C. for I hour ; live steam for 5 minutes ; 5 per cent acid carbolic for 12 to 15 hours ; 1 per cent corrosive sublimate for 2 to 3 hours. Direct sunlight diminishes their virulence and ultimately destroys them, otherwise they may remain virulent for years (in one case, 11 years). Are best formed at 370 C, but also form at 200 C. in 8 to 10 days. Habitat. — Soil, street dust, horse-dung. Pathogenesis. — In man : mostly from punctured wounds. The bacillus remains at the local site, but the toxins are carried to the nerve cells of the motor horns of the spinal cord, and of the motor ganglia of the brain. The manner of transmission is believed to be by absorption through the end-plates of the motor nerves in the muscles, and thence via the axis-cylinder processes to the respective nerve cell. The toxins have been shown to have no effect on the motor or sensory endings of the nerves, but solely as an exciter of the nerve cells concerned in reflex action in the cord, pons, and medulla. The affinity of the toxins for the nervous system varies in different animals ; in the guinea-pig it is its chief affinity, whereas in the alligator it shows no affinity, and intermediate degrees exist. Section of a nerve, e.g., the sciatic nerve, followed by injection of the toxins into the muscles supplied by that nerve, prevents the toxins reaching the spinal cord ; but if the nerve below the section be cut out and introduced into. a mouse, the animal will die of tetanus. Similarly, infection of one side of the cord passes, when the dose is 314 PUBLIC HEALTH BACTERIOLOGY large, to the other side via the commissure, and up the cord to the higher centres. The latter extension can be prevented by section of the cord. Lately, in India, the relation between subcutaneous or intramuscular injections of quinine (given for malaria) and the production of tetanus has been worked out. Such quinine injections are apt to have a destructive action on the tissues, and the foci of dead tissue produced serve as suitable anaerobic media for the growth of tetanus spores. The latter are believed to reach these foci by absorption from the bowel. This explanation will also probably serve for the Mulkowal outbreak (1902), when 19 persons developed tetanus (out of 107 injected) after inoculations of Haffkine's plague prophylactic. In India, tetanus spores seem to be present in the bowel in a considerable proportion of the natives. Toxins. — Broth cultures grown anaerobically are usually highly toxic to animals, 0-000005 c.c. (0-00V0 o) or ^ess Demg fatal to a mouse of 10 grm. weight. Fatal dose for a man is given as 0-23 mgr., equal to 0-003 of a grain, or ^J^. The maximum yield is given in 10- to 14-day-old cultures. After this it rapidly deteriorates. This also happens after separation from the bacilli by filtration, and in a few days it may have only TiF of its original power. Von Behring, who first noted this change, attributed it to the action of light, temperature, and especially oxygen, on the toxins ; and so such filtrates should be kept, covered with a layer of toluol and in a dark cool place. Exposure for a few minutes to 650 C. destroys it, as do 20 min. at 6o° C. and 1-5 hour at 550 C. Drying has no effect apart from temperature. It can be precipitated by over-saturation of the solution with ammonium sulphate, and thoroughly dried and stocked in vacuum tubes, together with anhydrous phosphoric acid, it may be preserved indefinitely without deterioration. The ordinary effects of the toxin are attributed to " tetanospasmin." Besides this, a substance named " tetanolysin " which has the power of destroying the red corpuscles of various animals, was discovered by Ehrlich. Tetanus toxin can be fully neutralized by mixing it with brain substance. Tetanus toxin is peculiar in that, after introduction into an animal's body, a definite incubation period occurs SPORING BACILLI 315 before symptoms arise. In the guinea-pig this is thirteen to eighteen hours, and in the horse five days. It is shorter after intravenous injection, probably through getting more quickly to the nerve centres. Crocodiles are resistant to tetanus toxin. Immunity. — Produced by injection of filtered toxin in increasing doses. At Elstree, the serum-producing department of the Lister Institute, London, the horse is immunized by the injection of 0-5 c.c. filtered toxin -f 0-5 c.c. of Lugol's solution of iodine (1-300), repeated at intervals of ten days, gradually increasing the dose until 10 c.c. of unreduced toxin are given. The iodine solution neutralizes the toxin to some extent. The serum of such an immunized animal is antitoxic ; but the effect of its injection into an infected animal is not so good as is the case with diphtheria antitoxin, because the tetanus is mainly bound to the nervous tissue and is thus less susceptible to the action of the antitoxin. Von Behring believes that there is no hope of its being useful after symptoms have existed for 30 hours, but MacConkey and Green say that if much larger doses were used better results would be got in the human subject, comparable with those reported in horses. The serum is standardized so that 1 grm. will protect 100,000,000 grm. weight of mouse (v. Behring), or 1,000,000,000 grm. weight (Pasteur Institute). Of this 100 c.c. are advised to be injected subcutaneously, and in the case of the first, repeated. The argest dose that can be comfortably given at one spot is 20 c.c. Intravenous injection is said to give better results than subcutaneous injection. The serum is warmed to the body temperature and slowly introduced into an arm vein, 10 to 20 c.c. every few hours. Intracerebral injection has also been practised, but with no better results. Prophylactic doses (10 c.c.) are advised as a routine practice in ragged, bruised, and punctured wounds, especially if soiled with material likely to contain tetanus spores. The dose is given without unnecessary delay. In U.S.A., in 1903, out of 4449 Fourth of July accidents, 406 were followed by death from tetanus, while in 1907, only 62 tetanus deaths arose from 4413 accidents, and much of the decrease is attributed to the early use of a 316 PUBLIC HEALTH BACTERIOLOGY prophylactic dose of antitoxic serum. In veterinary practice, prophylaxis has been used with great success. Search for B. Tetani in a Suspicious Wound. — (a) Microscopic examination of films for drumstick shapes. (b) Cultivation in deep stabs in glucose media for forty-eight hours. (c) Inoculation into mice and guinea-pigs. A loopful of the discharge into the root of the tail of a mouse will soon give rise to characteristic symptoms if B. tetani be present. From cultures Kitasato uses splinters of wood dipped in same, then heated to 8o° C. for i hour, which kills all non-sporing organisms and destroys any toxin developed. A splinter is introduced subcutaneously, and if death results it is from the spores which it carries. Bacillus Botulinus. — Found in "meat-poisoning" by raw ham by van Ermengem in 1896. The symptoms of the illness resembled those following sausage (botulus) poisoning, frequently met with in Germany from the ingestion of raw sausage. The symptoms follow at the earliest in twelve to twenty-four hours after the eating of the food. They are due to the action of a soluble toxin on the medullary centres, causing dysphagia, salivation, dilated pupils, and respiratory and cardiac distress. Fever is usually absent and consciousness is retained. B. botulinus is a large bacillus 4 to 9 micra long x 0-9 to 1-2 micron thick, with rounded ends. It is slightly motile, and has four to eight peritrichal flagella. It forms oval spores at one end, rather thicker than the bacilli, and these show slight resistance (1 hour at 8o° C). Strict anaerobe ; liquefies gelatin ; Gram-positive. Cultures. — Characteristic growth on glucose-gelatin plates : round, yellowish, transparent colonies, composed of coarse granules which (under a low power) show a streaming movement, especially at the periphery. Forms gas in glucose, but not in lactose nor sucrose. Milk is not coagulated. All cultures have a sour odour. The toxin is closely related in its action to the toxins of diphtheria and tetanus. The bacilli do not seem to multiply in the body, but the toxin is absorbed from the alimentary canal and produces the symptoms. The infected ham or sausage SPORING BACILLI 317 shows the bacilli in large numbers between the fibres. Thorough cooking destroys the toxin (not so in Gaertner meat-poisoning). The meat may be without any signs of ordinary decomposition. An antitoxin has been produced by Kempner, who also cultivated the bacillus from the intestine of the pig. Botulism is a dangerous affection, ending fatally in 25 per cent of those attacked. Bacillus of Malignant CEdema. — Discovered by Pasteur in 1877, in guinea-pigs inoculated with putrefying animal tissues. Gaffky found it in the upper layers of the soil of gardens and in dust. It is widely distributed in nature, and has been found in the intestine of animals and man. Its spores are very resistant, and are placed in the centre or near it. They are oval-shaped and slightly bulge the bacterial body. Spore formation occurs above 200 C. and is usually well seen in forty-eight hours at 370 C. The guinea-pig, rabbit, sheep, and goat are susceptible to inoculation ; the ox is immune to experimental infection, but has contracted the disease by natural channels. The bacillus is long (4 to 9 micra) and rather thinner than the anthrax bacillus, being 0-9 to 1-2 micron thick. The bacilli have somewhat rounded ends, and at times form threads. They are motile, have numerous peritrichal flagella, and are strict anaerobes. They stain readily by the usual aniline dyes ; they are Gram-negative. Cultures. — They grow best in the presence of glucose, and produce a heavy, putrid odour. They liquefy gelatin, and in deep stab show bubbles of gas around the colonies. They grow rapidly in deep stab in glucose agar, and here also gas forms and usually splits the medium. In broth, there is general clouding but no pellicle ; a granular sediment forms. In milk, slow coagulation is produced. On blood serum, growth is luxuriant. On potato, growth readily occurs. Inoculation of a guinea-pig (subcutaneously) produces death in twenty-four to forty-eight hours. There is an intense inflammatory oedema around the site of puncture and injection, which gradually extends to the surrounding tissues. The skin and subcutaneous tissues are infiltrated with a reddish-brown fluid, are softened, contain bubbles of gas, and are in places gangrenous. The superficial 318 PUBLIC HEALTH BACTERIOLOGY muscles are also involved, and have a putrid odour. The internal organs are congested and show parenchymatous degeneration. The spleen is soft, but not much enlarged. Immediately after death bacilli are not found in the blood or internal organs ; but thereafter the bacilli rapidly spread into the blood and organs. This account applies to the mixed infections (garden soil) ; in pure infection, little gas and odour are formed. Toxins. — A small amount of soluble toxin is formed, and filtrates of cultures in fluid media produce the same sym- ptoms (if used insufficient quantity) as the bacilli themselves. Chamberland and Roux, in 1887, produced immunity in guinea-pigs by the injection of the toxin, obtained by filtration or by sterilization of cultures by heat, or by filtra- tion from the serum of animals dead of the disease. Pasteur called the disease " septicemic," but it is not a true septicaemia like anthrax, in which the bacilli invade the blood and organs. It is a rare disease, occurring in man after traumatism. Quarter-Evil. — A disease of cattle, sheep, and goats, called by the Germans, " Rauschbrand," and by the French, " charbon symptomatique." It has never been observed in man. Infection takes place by some wound of the surface, and occasions inflammatory swelling with bloody oedema and emphysema of the tissues ; the affected part becomes greatly swollen, and of a dark, almost black colour. The bacillus is found in the inflamed tissues and in small numbers in the blood of internal organs. It closely resembles the B. cedematis maligni, but is somewhat thicker and does not form such long threads (filaments). The spores also are more bulging and nearer the end of the bacilli. An acute disease of sheep in Northern Europe, called " braxy," is associated with the presence of a very similar, if not identical anaerobe. Active and passive immunization of sheep and goats and cattle are practised. B. Enteritidis Sporogenes was first isolated by Klein in 1895 from diarrhoeal stools. It was afterwards found in infantile diarrhoea and summer diarrhoea, and as a constant inhabitant of sewage. It is slightly motile, with a tuft of flagella at one pole (lophotrichal), i-6 to 4-8 micra long by o-8 micron thick ; easily stained ; Gram-positive ; gelatin- SPORING BACILLI 319 liquefying, and produces acid and gas in bile-salt glucose media and in peptone water + glucose or mannite. It forms a spore nearer one end. Its growth in milk is highly characteristic, and this medium is commonly used for its isolation. Method. — A small quantity of the suspected material is inoculated into sterile milk (" whole milk "), using at least 15 c.c. of the medium. Heat for 10 minutes at 8o° C. to destroy all non-sporing forms, cool the tube, and incubate anaerobically for twenty-four to thirty-six hours. If the casein is precipitated and torn into irregular masses, with a moderately clear whey and abundant gas formation, the result is positive, but it is desirable to verify by animal inoculation. (In the examination of water and milk, the result is observed after two days' incubation.) The culture has a smell of butyric acid, and numerous bacilli are found in the whey. If 1 c.c. of the whey be injected into a guinea-pig, the animal becomes ill in a few hours, and dies in twenty-four hours. At the point of inoculation the skin, subcutaneous tissues, and sometimes the adjacent muscles, are green, gangrenous, ill-smelling, and cedema- tous ; there may be gas formation. This pathogenic test serves to distinguish the B. enteritidis sporogenes from the B. butyricus of Botkin, which otherwise closely resembles it. B. Aerogenes Capsulatus. — First observed by Welch in 1891, and obtained from the intravascular blood in a case of ruptured aortic aneurysm. The post-mortem took place six hours after death, and attention was called to the blood by the presence of gas-bubbles throughout the vessels. It was fully described by Welch and Nuttall in 1892, and in 1893 Fraenkel independently described (under the name of B. phlegmonis emphysematosa?) a bacillus, now considered to be identical with the B. aerogenes capsulatus. Klein's B. enteritidis sporogenes is believed by some to be the same organism, or a closely related one. B. aerogenes capsulatus is widely distributed in nature, being found in soil, dust, brackish water, and in the normal intestinal tract of man and animals. In size it is not unlike anthrax bacillus, but is more variable in length and somewhat thicker. The bacilli are generally 320 PUBLIC HEALTH BACTERIOLOGY single or in short chains, and are shorter and thicker in cultures. Chain formation seems to occur in the blood chiefly, and never in artificial culture. Welch regards this as an important distinction from anthrax bacilli. When recovered from the body fluids it possesses a capsule. Each bacillus forms one spore, which may be central or excentric. Anaerobic ; non-motile ; non-flagellar ; Gram-positive ; gelatin-liquefying (in most), and forming acid and gas in glucose, lactose, and saccharose, but not in mannite. In milk the reaction is similar to that described under B. enteritidis sporogenes. It is highly pathogenic to guinea- pigs but not to rabbits. Isolation. — Make a suspension of the suspected material (faeces, etc.) in sterile salt solution (i c.c. in 5 c.c). Thoroughly emulsify and filter through a sterile paper, and inject 1 to 2 c.c. of the filtered suspension into the ear vein of a rabbit. After 5 minutes, kill the rabbit and place its dead body in the incubator (370 C.) for 5 or 6 hours. At the end of this time the animal is usually found tensely distended with gas, and post mortem gas bubbles will be found throughout the body, most characteristically in the liver, where isolated bubbles are found on the surface. From the bubbles, smears and cultures may be taken. Identification is made from its morphology, capsule, non- motility, and gas formation. In man, infection usually follows traumatism. Distinction from B. enteritidis sporogenes : non-motility, non-flagellar, not fermenting mannite. Muir and Ritchie state that it is non-gelatin- liquefying and non-pathogenic to guinea-pigs, but American authors . describe it as above. Summary. Bacillus Motility Flagella Gram. Gelatin- liquefying Spore Tetani + peri- trichal + + end (drumstick) Botulinus + + + near end Malignant oedema + ., — + central Quarter-evil + " — + near end — - racket shape Enteritidis sporo- + lopho- + + central or near genes trichal end Aerogenes capsu- — — + + ,, ,, lars . . \> . . 1 CHAPTER XV. SPIRILLA. SPIRILLUM CHOLERA ASIATICS. The cholera spirillum was discovered by Koch in 1883 in the defalcations of sufferers from cholera. It is also called the " comma bacillus " and the " Vibrio cholerae." Description. — Short, slightly curved rods, 1-5 to 2 micra long by 0-5 micron thick. Ex. C In pairs, may form an S-shape, thus *y Actively motile, and swim like fish in lines, thus 5 5 = 5 . Possess one flagellum, situated at one end (monotrichal) . Non-sporing ; not phosphorescent ; Gram-negative. Markedly aerobic, but can grow anaero- bically. Optimum temperature 370 C. ; growth usually ceases at 160 C. Gelatin-liquefying. Give nitroso-indol reaction with sulphuric acid within twenty-four hours. Culture. — Grows readily on all usual media, but better if alkaline, and except on potato even at room temperature ; characteristic on gelatin plates and in broth. On gelatin plate : minute whitish points in twenty-four to forty-eight hours ; surface when magnified is coarsely granular and furrowed. Liquefaction follows and the colony sinks, showing a ring around. In gelatin stab : liquefaction begins at the surface, with gradual formation of a funnel of liquefaction. In broth : rapid clouding occurs with wrinkled pellicle on top, composed of spirilla in a very actively motile condition. In milk : growth but no visible change. In peptone water : rapid growth with production of indol, and reduction of nitrate to nitrite, hence a few drops of pure sulphuric acid will give a red colour — the so-called cholera-red reaction. Also given in broth culture, and in both in twenty-four hours, owing to rapid growth. Not absolutely specific, as it is given also by Sp. Metch- nikovi. Blood serum is rapidly liquefied. In sugar media : no gas is formed, but acid with glucose. Does not pro- duce haemolysis, though very similar species do. Does not multiply in water. 21 322 PUBLIC HEALTH BACTERIOLOGY Staining. — Readily with usual stains, best with Loeffler's methylene-blue and weak carbol-fuchsin. Loses stain by Gram's method. Resistance. — Not great. Killed in ten minutes at 6o° C. ; on drying, in two hours. Mineral acids, I in 5000 to 1 in 10,000, destroy it in a few minutes. The gastric juice con- tains 2 parts of HC1 per 1000 ; hence it is killed by gastric juice, but can flourish in intestine. Freezing kills it in three to four days. Agglutination — Is shown by serum of cholera con- valescents in dilutions of 1-15 to 1-120. Present eight to ten days after attack, most marked twenty-eight days after, and gradually diminishes. Has been noted as early as first day of disease. Pathogenesis. — For man : has been established by laboratory accidents. Does not invade blood ; immense numbers in stools ; in rice-water stools, loosened epithelial cells loaded with vibrios. Not in urine. Infection by mouth. Disappears from stools in two to three days. Cholera carriers : healthy persons whose faeces contain virulent cholera spirilla. Mostly spread by water, fomites, ringers, flies. For animals : not established, though vibrios on teats of suckling mother have infected young, with choleraic symptoms. But as this result or similar ones have been given by other vibrios, specificity cannot be founded on this test. Intraperitoneal injection in guinea-pigs is followed by general symptoms, with abdominal distension, subnormal temperature, and profound collapse. There is peritoneal effusion which may be almost clear, or with flakes of lymph. There is little tendency to invade the blood-stream, and the symptoms are mainly due to an intoxication. It is not pathogenic to pigeons. Toxins. — Filtered cholera cultures have as a rule little toxic action ; hence it is inferred that little soluble toxin is formed, but mostly endotoxin. Results at present are conflicting. Pfeiffer's Reaction. — If cholera spirilla are injected into the peritoneum of an immunized guinea-pig, they first lose their motility, then swell up and crumble into frag- ments, which finally melt away and disappear. This lysis is also manifested in a test tube in a mixture of serum SPIRILLA 323 plus vibrios. Or, inject a mixture of one loopful (2 mgr. of recent agar culture and 1 c.c. of broth containing o-ooi c.c. of anti-cholera serum into the peritoneum of a guinea- pig. Remove some fluid at regular intervals and examine. If above reaction is got, then said to be positive, and in case described, organism is proved to be true cholera spirillum. If the latter is used against an unknown serum, then the anti-power of the serum can be determined and, if positive, by using various dilutions, the bacteriolytic power. Immunization. — A guinea-pig is easily immunized by repeated injections of non-fatal doses of dead spirilla ; later small doses of living organisms may be used. A high degree of immunity is thus developed, and the blood serum of such an animal (anti-cholera serum) when injected into another guinea-pig has marked protective power. This is not due to any antitoxic substances, but to antibacterial power. Cholera-immune serum is thus bacteriolytic, not antitoxic. This power is specific, and does not apply to other closely related organisms. Hafjkine's Vaccine. — First vaccine : Attenuated virus by long cultivation at 390 C. or by other methods. Second vaccine : Given five days later, of virulent virus (by passage through guinea-pigs). Both given sub- cutaneously. Lately has given only one, and that the " virus exalte." General conclusions as to efficacy : (1) Protective effect of anti-cholera vaccine commences soon after operation and increases rapidly for first four days, and lasts fourteen months, after which it diminishes and completely disappears. Larger doses cause longer effects. (2) During period of its activity, the number of cases among vaccinated is one-tenth of number among others. (3) Mortality among those attacked differs but little, and the course of the disease is not affected by the previous inoculation. Isolation.— 1. From Faces. — Make pre-culture in peptone water. (a) Inoculate peptone water in Erlenmeyer flasks (1 loopful in each), (b) In eight hours, if a film appears or not, make hanging drop from surface, and if you get fish trains, dry and stain to see if form is typical, (c) Subculture from film on gelatin plates, and smear over agar plates. 324 PUBLIC HEALTH BACTERIOLOGY (d) After eight to ten hours examine any colonies, and if pure cultures, plant out as follows : (i) Peptone and salt solution : in twenty-four hours at 370 C, turbid, and gives cholera-red ; (ii) Gelatin plates : characteristic colonies with irregular margins ; (hi) Gelatin stab : typical funnel- shaped liquefaction ; (iv) Agar slope : growth in twenty- four hours at 370 C. must give with anti-cholera serum, agglutination and Pfeiffer's test ; (v) A portion of colony should be examined for typical microscopical appearances. Make films and hanging drops direct from stools. Dunbar diagnoses from two hanging drops, one having added to it an equal quantity of 1-50 normal serum, the other an equal quantity of 1-500 anti-cholera serum. Cholera organisms retain their motility in the first instance, but lose it and agglutinate in the second. The hanging drop is mounted from peptone water in which a piece of mucus has been broken up. 2. From Water. — Keep 10 per cent peptone water sterilized. Take 900 c.c. of suspected water, and add 100 c.c. of strong peptone water. Divide into ten flasks, each containing 100 c.c. Incubate at 370 C. In eight to twelve hours make a film and hanging-drop preparation from the surface of each flask. From those flasks showing most similar forms, make subcultures, proceeding as above. If a spirillum conforms to all the above tests, it is probably the true cholera vibrio, but it must be remem- bered that a certain number of spirilla, although agglutina- ting to some extent with cholera serum, are sharply differentiated by being multiciliated and hemolytic. SPIRILLA OTHER THAN THE CHOLERA SPIRILLUM. (Often present in water but not necessarily pathogenic). Sp. Metchnikovi — Is found in a disease resembling fowl cholera in the faeces and blood. It is practically identical with the Sp. cholerae, having a single polar flagellum. Culturally, it fluidifies gelatin twice as rapidly, and grows slightly more luxuriantly. It is sharply distinguished, however, by being very pathogenic to pigeons (doves), SPIRILLA 325 whereas Sp. cholerae is scarcely so. It is negative to Pfeiffer's test, but gives the cholera-red reaction. Sp. Massaua (Massowah) — Was isolated in a small epidemic of cholera and accepted as the true spirillum, but further study showed that it was negative to Pfeiffer, was very pathogenic to pigeons, and possessed four flagella. Sp. of Finkler and Prior— Was isolated first from faeces of a case of cholera nostras, and has since been found in water. Morphologically, it is like the Sp. cholerae, though thicker in the centre and more pointed at the ends. It does not give the cholera-red reaction, and liquefies gelatin very rapidly, showing no bubble-like appearance. Grows well on potato, and is negative to Pfeiffer. Sp. Aquatilis of Gunther — Was found in Spree water, and closely resembles Sp. cholerae, but young colonies have a smooth rim, and it does not give cholera-red reaction, and is negative to Pfeiffer. Does not grow on potato. Sp. Danubicus. — Cultivated from canal water. Does not give Pfeiffer, and colonies are different ; otherwise it closely resembles the cholera spirillum. Sp. Deneke — Also called Sp. tyrogenum, was isolated from butter and old cheese. It closely resembles the Sp. cholerae, but is thinner and smaller ; growth in gelatin similar but more rapid, and does not give the cholera-red reaction. It is very feebly pathogenic, and is usually regarded as a harmless saprophyte. Sp. Phosphorescens — Gives luminous cultures. CHAPTER XVI. SPIROCHETES. The diseases produced by spirochetes are now referred to as spirilloses or spirochetoses, and fall into (following Muir and Ritchie) two main groups : — (i) The human spirillar fevers, and the corresponding affections of various animals ; (2) Syphilis and yaws, and the ulcerative and gangrenous conditions apparently caused by spirochetes (e.g., Vincent's angina). In the first group, blood infec- tion is the rule, and the organisms are, in most cases if not in all, transmitted by blood-sucking ecto-parasites. In the second group, the organisms are primarily tissue parasites, and later show blood infection, and are mainly spread by direct contact. As regards general morphology, staining reactions, and conditions of growth and culture, the various spirochetes present common characters, and their classification with bacteria or protozoa is still a matter of doubt. Spirillum Obermeieri or Sp. of relapsing fever (Ober- meyer, 1873) — Is now usually regarded as a spirochete, and known as the Spirochaeta recurrentis. It is found in the blood of patients suffering from " relapsing fever " from shortly before the onset of the pyrexia until shortly before the crisis, and similarly in the relapses. The relationship of the organism to the disease has been proved by the injection of spirochetes into the blood-stream causing the typical attack, both in the human subject and in monkeys ; also in white mice and rats, but in these and in monkeys, relapse is rare. Sp. Obermeieri is a delicate spiral thread, 7 to 9 micra long by about 1 micron thick, but the size varies from one-half to nine times the diameter of a red blood corpuscle (7 micra). The windings like- wise vary from 4 to 10 or more. It stains with watery basic aniline dyes, somewhat faintly, but best with the Romanowsky stains. It shows a homogeneous cell body SPIROCHETES 327 or a few granules, but no division into segments. It is Gram-negative. Flagella have been noted. Spirochaeta Vincenti— Is a delicate spiral-shaped organism, said to be often found in the mouth as a simple saprophyte. It was described by Vincent in 1896, in an inflammatory lesion of the pharynx, since spoken of as Vincent's angina, in association with a fusiform bacillus of large size (3 to 10 micra by 0-5 to o-8 micron). The curvature of the spirochetes is irregular and the number of curves variable. The relationship of these organisms to the disease is still obscure. THE MICRO-ORGANISM OF SYPHILIS. This is variously called the Spirochaeta pallida of Schaudinn and Hoffmann, the Treponema pallidum, and the Microspironema pallidum. It was discovered by the two observers named in 1905, in the primary sore and in the adjacent lymphatic glands. It has since been demonstrated in numerous lesions and in the blood, and in congenital syphilis. It has been found in large numbers and in pure culture in the lungs, liver, spleen, pancreas, and kidneys ; and in a few cases in the heart muscle. It has also been found in the roseolar spots in the disease, and in blister fluid in an infected person. This shows how the disease can be spread (as at times it has been) by vaccine fluid. In the bubo, other spirochaetes are usually found in association with the Spirochaeta pallida. These are much thicker, less undulated, more retractile, and more deeply staining. They are spoken of as Spirochaeta refringens. These are said to have an undulating membrane (like the trypanosomes) but no flagella. The term treponema is now reserved for a genus with no undulating membrane, and flagella of some sort at their extremities. To this group the organism of syphilis belongs, and is hence now referred to more strictly as the Treponema pallidum. The Treponema pallidum is an extremely delicate spiral organism, very slender, about 6 to 14 micra long by less than 0-5 micron thick, showing about a dozen very 328 PUBLIC HEALTH BACTERIOLOGY regular spiral turns close together, the whole resembling a fine corkscrew. It has a flagellum at each extremity, but no undulating membrane. It multiplies by longi- tudinal division, the initial stage being shown by the splitting of the flagellum at one end. It can be demon- strated in the living state in a hanging drop, or a ringed-in cover-slip, cutting down the light to a minimum, or better, by using dark-field illumination. In smears, it can be stained by Giemsa's method, of which there are several modifications. A more rapid and simple method is by using India ink. A loopful of secretion from a chancre is mixed with a loopful of ink (Gunther & Wagner's liquid pearl ink), and the mixture made into a smear as for blood. Dry in the air, and examine with an oil-immersion lens. The treponemata appear as white spirals on a dark back- ground. In tissues, ordinary methods do not stain the organisms. Levaditi's method is commonly used, and consists in fixing in formalin for 24 hours, washing out the formalin with water, the traces of which, which might con- tain formalin, being removed with alcohol ; soak in silver nitrate solution for from three to five days, wash and soak in pyrogallol-formalin solution ; wash, dehydrate, imbed in paraffin, and section. A shorter method has been devised. The evidence of its pathogenicity is derived from its constant presence in the lesions of acquired and congenital syphilis, and in that it has been communicated to monkeys, producing typical syphilitic course and lesions, from which the treponema was recovered in 70 per cent of the cases examined. It has not yet been successfully cultivated in a pure state, or in that case complement fixation by a pure culture might be an additional proof. It is not regularly recovered from tertiary lesions, which is not surprising. This is analogous to tuberculosis, in which the tubercle bacillus is often not demonstrable by ordinary methods in the chronic lesions. Treponema pallidum does not pass through a filter. Wassermann Reaction — Is now regularly used in clinical diagnosis. It is described on page 205 ; its value is discussed on page 209. In a recent research by Calmette, Breton, and Couvrer, it has been practically applied to the diagnosis of syphilis in the newly born child, SPIROCHETES 329 with a view to securing early treatment in those cases which would not be diagnosed on ordinary clinical grounds. The mode of procedure was to examine the placental blood taken at the time of delivery, before ligature of the cord. Out of 103 such samples, in 16 a positive result was obtained. Out of the 16, no evidence was got of syphilis by clinical examination or history in the parents or child in 8 cases. Of the remaining 8, there was definite evidence in the parents or child in 5 cases ; and in one other the mother had previously borne an anencephalic monster. In every case in which the child showed signs of syphilis at birth, the mother's blood gave a positive reaction. Immunization. — Though one attack of syphilis usually protects against another infection beginning with a chancre, no success has yet been obtained in attempts to produce either active or passive immunization. Yaws. — A disease resembling syphilis, and at times regarded as identical with it, which prevails endemically in the West Indies, Brazil, Fiji, Ceylon, the East Indies, and different parts of Africa. It is called variously yaws, fram- bcesia, bubas, koko, paranghi, and pian. It is highly con- tagious, but is not hereditary or congenital. It is now believed to be due to Treponema pertenue, first described by Castellani in 1905 as Spirochaeta pertenuis, which resembles the Treponema pallidum closely. CHAPTER XVII. YEASTS AND MOULDS. Yeasts and moulds are grouped in the class of fungi to which the bacteria also belong. They are distinguished from the latter (and from one another) by their mode of reproduction. From the bacteria they also differ in being much larger, as a rule. Their biological requirements are also, generally, much less exacting. Between these groups and the average bacterium, the space is bridged by some forms called the higher bacteria, which resemble the moulds in showing branching. Such are actinomyces (which has been considered immediately after B. tuberculosis), the streptothricae, etc. These are often grouped as trichomycetes, which is regarded as a subdivision of the true moulds. The whole subject is at present uncertain and confused. Foulerton, in his Milroy Lectures (Lancet, 1910, Vol. i., p. 551 on) urges the view that the micro-organisms variously called tubercle bacilli, actinomyces, cladothrix nocardia, oospora, and strepto- thrix, belong to one family of moulds or hyphomycetes. In clinical medicine and pathology the term " mycoses " is used (following Virchow) to denote all the affections produced by filamentous and budding fungi, and this term associated with the seat of the lesion has given rise to such terms as dermatomycosis, otomycosis, etc. On the other hand, such terms as actinomycosis, saccharomycosis, blastomycosis, aspergillosis, sporotrichosis, etc., are used. All the members of these groups may be considered as facultative parasites, parasitic life being unnecessary to their cycle of evolution, as their proper existence is a saprophytic one. The parasitism is, in their case, simply a phenomenon of adaptation. Being destitute of chlorophyll, they do not need light, and grow luxuriantly in the dark. They vary much in their temperature requirements, a few growing well at YEASTS AND MOULDS 331 human body-heat, some at 300 to 330 C, and most at air temperatures. Growth at temperatures higher than the optimum, in certain media and anaerobically, results in the production of pleomorphic forms. Most of them die when deprived of air or oxygen ; a few are anaerobic. Moisture is absolutely necessary. They grow readily on organic matter of all kinds. The natural media commonly used in their study are bread, sterilized milk, beer wort, potato, carrot, decoctions of fruits, etc. As these media are vari- able in their composition from time to time, and the problem of pleomorphism has to be faced, artificial media of definite composition and reaction are preferred in scientific work for giving comparable results. Growth is best on solid media, standardized to an acid reaction of -f 2 per cent (+20 per litre). YEASTS. The yeasts are fungi characterized by the mode of multiplication known as " budding " or " gemmation' ' or asymmetrical fission, and are hence called blastomycetes. From their action in fermenting sugars they have also been called saccharomycetes. Their botanical position as a separate group is not well established, as a large number of intermediate forms relate them closely to the moulds. The usual yeast cell is round or oval in shape, 10 to 20 micra long by 5 to 15 micra across, and occurs singly or in short chains. Each cell is bounded by a cell-membrane composed of cellulose, and of such a thickness (0-5 micron) that it shows a double contour. Within the membrane is con- tained the protoplasm, in which is a large number of granules, globules, and vacuoles, and in old cultures a nucleus is sometimes seen. When budding, the mother cell throws out a small globular process, which gradually enlarges until it attains nearly the same size as the parent cell. By a gradual narrowing of the isthmus between the mother and daughter cells, the daughter cell finally becomes free. In addition to this mode of reproduction, most yeasts can form spores called " ascospores." This takes place when there is a lack of nourishment or where the conditions of life are otherwise unfavourable. These spores are 332 PUBLIC HEALTH BACTERIOLOGY formed by endogenous cell-division, and the usual rule is for the protoplasm of one cell to divide into four spores, each with its own cell-membrane, the original cell- membrane persisting and serving as an envelope enclosing the spores. Yeasts grow more slowly than bacteria, and are hence more difficult to isolate from mixed cultures on the ordinary media. Once isolated they are kept alive by subculture every 2 to 3 months. On glucose agar or plain agar, colonies appear in 3 to 4 days as minute glistening white spots. In stab, the growth is all at the top, forming a heaped-up creamy layer on the surface of the medium. In broth, a stringy gelatinous growth is formed. Growth takes place on gelatin, which is not liquefied. On potato, growth is more rapid. The cultivated yeasts used in brewing and baking processes are capable of fermenting various sugars. This action is due to certain ferments or enzymes elaborated by the yeasts. Two of the enzymes are diffused into the medium in which growth is taking place ; one remains closely bound to the yeast cell, and was only isolated by Buchner by rupturing the living yeast cells under great pressure, filtering, and centrifugal- izing the filtrate. This filtrate was found to have the power of fermenting glucose and laevulose into alcohol and carbonic acid gas. This endo-enzyme is called " zymase." C6H1206 = 2-C2H,-OH +>C02 The other two soluble enzymes are " invertase " and " maltase." The former inverts cane sugar or saccharose into invert sugar (glucose -f- laevulose), and so renders it susceptible to the action of the " zymase." " Maltase " acts on malt sugar, changing it into glucose, which is then acted on by the " zymase." C,,H22Ou + H20 = C6H1206 + C6HH06 Saccharose. Glucose -f- laevulose. ClaHMOn + H20 = C„HlaOa + QHl206 Maltose. Glucose -f- glucose. Saccharomvces cerevisise. — This is the name applied to the yeast in common use by brewers and bakers. YEASTS AND MOULDS 333 It consists of round or oval cells containing a clear fluid and no granules. It is not used older than one week's growth, after which time granules appear in the cells. When budding very rapidly, delicate mycelial threads are formed. It forms ascospores at 25 ° C. in thirty hours, and at 120 C. in ten days. In brewing, yeast is added to the beer wort (cooled to 160 C), and fermentation takes place. A brownish-yellow scum forms on the surface, bubbles of gas (C02) escape, forming a foam, and alcohol is formed in the liquid. This is the " high " fermentation with " top " yeast, and takes place in several days. In some breweries " low " or " bottom " yeast is used, and the fermentation is conducted at 50 C. The yeast cells sink to the bottom as they are formed, and the whole process takes a much longer time (fourteen days). In baking, the yeast converts the starch into sugar, and then the latter into C02 and alcohol. The gas breaks up the glutin into thin-walled cells. The subsequent " baking" or heating kills the ferment, and drives off the CO 2 and alcohol. Wild yeasts are usually termed torulcc. They have oval or spherical-shaped cells, do not produce ascospores, and have only feeble fermentative powers. Some of them have been found to produce a true mycelium. Torula rosea (Saccharomyces rosaceus) is a pink torula, which, growing on gelatin, agar, etc., produces raised masses with a polished pink surface, similar to a piece of coral. Microscopically, it shows rounded or slightly oval cells, 5 to 8 micra in diameter, and containing a delicate yellow pigment, which in the mass gives the pink shade. Torula niger (Saccharomyces niger) grows on gelatin as a black heaped-up mass, resembling a piece of black sealing- wax. On potato and bread paste it forms a dull sooty crust, and in milk a black crust. It is met with in the air. Pathogenic yeasts have been described in connection with (1) Multiple abscesses in bones, lungs, spleen, and kidney, and ending fatally (Busse) ; (2) An illness simula- ting diphtheria (Klein and Gordon) ; (3) Subcutaneous myxomatous tumours (Curtis) ; (4) Middle-ear disease (Maggiora and Gradenigo) ; (5) A lupus-like skin disease 834 PUBLIC HEALTH BACTERIOLOGY (Gilchrist) ; (6) An intraperitoneal tumour (Blanchard, Schwartz, and Binot), etc. MOULDS. The distinguishing feature of the moulds is their growth in long threads or filaments, with seed-bearing branches called hyphae. Each filament may be a single, simple, multinuclear cell, or a greatly branched one, or may be composed of a row of cells set end to end. The interlacing mass of threads is called the " mycelium." On this basis moulds are divided into two classes : (i) Phycomycetes, or those in which the mycelial threads consist of a single cell ; and (2) Mycomycetes, in which the mycelial threads are composed of numerous cells. The two groups also differ in that in the first, reproduction is sexual and asexual ; and in the second is by the asexual process only. All moulds prefer an acid to an alkaline medium, and hence are found attacking fruit preserves and similar substances. The spores of moulds are present everywhere, and in the air are in greater numbers than bacteria. The members of this group of fungi which we shall consider are : Mucor mucedo ; Aspergillus ; Penicillium ; Microsporon furfur ; M. minutissimum ; Sporotrichum Beurmanni ; Oidium albicans ; and the moulds of ring- worm. Mucor mucedo is the commonest mucor or "head" mould, and belongs to the class of single-celled fungi or phycomycetes. It is the common, white, cottony mould which grows on damp bread, rotten fruit, horse dung, etc. There is a finely branched mycelium from which project thicker unbranched hyphae. Near the end of these hyphae a septum forms, the terminal portion of the hypha swells, and in it numerous oval spores develop. The globular swelling produced is known as the " sporangium," and is enclosed by a capsule. It ruptures when ripe by the swelling of the gelatinous material in which the spores are imbedded. The end of the hypha projects into the spor- angium, and this part of it is called the " columella." This asexual form of multiplication is the more common, but sexual reproduction occurs under conditions not well defined. YEASTS AND MOULDS 335 In this form lateral branches (garnet ophores) grow out from two hyphae close to each other. These gametophores meet by the tips, fuse, and then by septa the central portion is separated off and becomes a " zygospore." The mature zygospore under suitable conditions enlarges and sends out a germ tube or hypha, on the end of which a sporangium may appear. Mucor grows on gelatin plate as round white colonies which soon cause liquefaction. In gelatin stab, it forms a dense white growth spreading over the surface, and sending down penetrating branches — sub- aerial hyphae ; others rise up vertically into the tube — aerial hyphae. Other mucors are : M. stolonifer (black mucor), M. spinosus (chocolate colour : has spines on the columella) . Aspergillus or "Knob" Mould. — This is a common form of mould, occurring on bread, cheese, oranges, etc. There are several varieties, A. glaucus (blue mould), A. niger (black mould), A. flavus (yellow mould), and A. fumigatus (green turning to grey). The mycelial filaments are composed of numerous rod-like cells joined end to end. They reproduce asexually. Hyphae arise from the mycelial network, and each hypha terminates in a knob-like expan- sion, the columella. The surface of the columella becomes studded with flask-shaped organs or cells called sterig- mata, and each of these forms spores or conidia, which remain attached in chains like streptococci. The result is a knob with radial projections composed of spores ; but having, unlike Mucor, no containing capsule. Aspergilli grow on gelatin as round white colonies very like those of penicillium. In a few days coloured points appear, denoting spore formation, being blue in glaucus, black in niger, etc. The gelatin is liquefied. In gelatin stab there is a dense felt-like growth (more pronounced than with penicillium), and later liquefaction. The pathogenic aspergilli include : — i. Aspergillus fumigatus, which has been found on the one hand in a malady simulating pulmonary tuberculosis, but not showing tubercle bacilli in the sputum (at times it is associated with the tubercle bacillus) ; and on the other hand, causing affections in the external auditory canal, the tympanic cavity, the nasal fossae, and in wounds. 336 PUBLIC HEALTH BACTERIOLOGY Such infections mostly occur in those engaged in handling grain, whole or crushed, either as transporters or as feeders of animals or fowls. Many of the cases are among bird fanciers, especially those keeping pigeons, which among birds are specially liable to aspergillosis. Birds and mammals can be fatally infected by intravenous inoculation with aspergillus spores. In birds, infection has been produced by inhalation of spores. In examining sputum for the presence of aspergilli, it is absolutely necessary that the examination should be made immedi- ately after expectoration, since the spores of such moulds may exist in the air in considerable numbers, and falling on to the sputum would germinate there. A film is made on a slide in the ordinary way, dried, fixed by heat or absolute alcohol, stained with carbol-thionin, and examined. The characteristic threads or filaments of the mycelium are seen among the pus cells. It is further necessary to verify the diagnosis by cultivating the fungus and noting its growth and morphology. This should be done on a special medium, such as Raulin's liquid medium, in which the aspergillus grows well. This is composed of water, 1500 grm. ; crystallized sugar, 70 grm. ; tartaric acid and ammonium nitrate, of each 4 grm. ; ammonium phosphate and potassium carbonate, o-6 grm. ; magnesium carbonate, 0-4 grm. ; ammonium sulphate, 0-25 grm. ; sulphate of iron, sulphate of zinc, potassium silicate, and manganese carbonate, of each o-p7 grm. Take a sufficiency in an Erlenmeyer flask, inoculate by dropping in a small piece of the sputum, and incubate at 370 C. In 3 to 10 days there grows a whitish meshwork, with branches bearing spores, green at first, but in a few days becoming smoke-black. The growth is examined microscopically, and its characters are studied. To verify its pathogenic action, an emulsion of the culture is injected into the ear- vein of a rabbit. The animal dies in several days with a generalized pseudo-tuberculosis. 2. Aspergillus repens, found in the auditory canal, producing a false membrane. 3. Aspergillus flavus, in chronic ear discharges. Penicillium, or " Pencil " Mould.— The blue-green variety of this form of mould, Penicillium glaucum, is the YEASTS AND MOULDS 337 most commonly occurring of all moulds. In this genus, the mycelial threads are septate or many-celled. Hyphse are given off, and from the end of each hypha, two or more short pencil-like branches arise, and these likewise give origin to other similar branches. These last, or further set of branches, produce spores or conidia, which, remaining attached, form a string of spores. The branches producing the spores are called sterigmata, and the inter- mediate branches the basidia or conidiophores. The result is not unlike an #-ray photograph of the arm, in which the humerus represents the hypha, the radius and ulna — two basidia (omit the wrist), the metacarpus — five sterigmata (say three from the radius and two from the ulna), each sterigma bearing spores — the phalanges. The spores are rounded in shape. Penicillium glaucum grows on bread paste, showing at first a white fluffy growth, becoming either green or blue, as the spores form. It sometimes is covered with little drops of dew-like fluid. On gelatin plates it grows as small round colonies of hair-like filaments, at first white in colour, but later greenish. The gelatin is liquefied. In gelatin stab a white fluffy layer or scum rapidly forms on the top, and descending branches run into the gelatin, as well as hori- zontal ones from the stab. The medium becomes bluish or greenish, and liquefaction takes place. Growths on agar and potato have similar characters. Penicillia have been described as the cause of chronic catarrh of the Eustachian tube, and of gastric hyper- acidity. Microsporon furfur — Is a mould first described in 1846, and found in the skin affection called pityriasis versicolor. It is composed of sinuous hyphae, 3 micra in thickness, showing right-angle branches. The spores are large, 3 to 5 micra in diameter, and are formed in a manner similar to that in penicillium. Pityriasis versicolor is seen in persons subject to profuse perspiration, who have been infected with the spores from the air or elsewhere. The fungus grows in the superficial layers of the epidermis, forming a yellowish' or coffee- and-milk coloured patch, usually seen on the chest or abdomen or back. Little or no discomfort is caused to 22 338 PUBLIC HEALTH BACTERIOLOGY the person affected. The diagnosis is easily confirmed by examining a scale in a drop of liquor potassas ; or the scale may be teased out on a slide in a drop of absolute alcohol, and then stained with eosin. The filaments and the large round spores are readily seen. Microsporon minutissimum is a mould described as the cause of dhobie's itch or erythrasma, which is a common affection in the tropics. It is mainly seen in the axillae, the scrotal region, the insides of the thighs, and the submammary folds. Like the Microsporon furfur, it lives a simple saprophytic existence in the epidermis, causing reddish-brown patches with an abrupt edge. When a scale is removed, washed with ether, teased out in acetic acid, allowed to dry, washed with alcohol, and stained with carbol-thionin, the fungi can be seen with the microscope as slender sinuous filaments, formed of short elements, very similar to bacilli, from destruction of parts of the filaments, or non-staining of these parts. Sporotrichum Beurmanni is a mould composed of a mycelium, the filaments of which branch in all directions. The hyphae are i to 2 micra thick, and at the ends of these oval spores are formed (3 to 5 micra by 1*5 to 3), singly or in grape-like clusters. Spores are also formed around the main filaments, or apparently so. The full life-cycle of the sporotrichon has not yet been worked out, and its exact classification is still a matter of doubt. It was first isolated by Schenk in 1898 from refractory subcutaneous abscesses in man. De Beurmann and Ramond rediscovered it in granulomata in the skin in 1903. Since then numerous cases have been reported in France, and lately two cases have been reported in this country (Ofenhein, Lancet, 191 1, Vol. 1, page 659 ; and Norman Walker and James Ritchie, British Medical Journal, 1911, Vol. 2, pages 1-5. The latter article is accompanied by a special coloured plate and a short bibliography). Sporotrichosis is a disease characterized by cutaneous and subcutaneous tumours, firm and indolent. These may ulcerate and discharge a viscid homogeneous pus of a yellowish-grey colour. The tumours have been in the past mistaken for those due to syphilis and tuberculosis, and pot- assium iodide and tuberculin injections or other treatment YEASTS AND MOULDS 339 administered. When iodide was given, cure was often effected, and so the diagnosis was apparently confirmed. The ulcers have similarly been treated as syphilitic, as lupus, and as simple pyogenic ulcers. In many cases there is a history of a minor injury, with a spread from this up the line of the lymphatics, with tumour formation and breaking down at various points en route. The lymphatic glands are not usually enlarged, the fungus probably not reaching them at an early stage. The affection is usually not a serious one, but there is little tendency towards cure if left untreated. Stimulating local treatment, together with the administration of large doses of potassium iodide (60 to 80 grains, or 4 to 5 grm. daily) is quickly followed by cure. The diagnosis is based on the direct examinations of scrapings, which are usually negative except for spores, oval and 3 to 5 micra long ; and by cultivation. Pus from an unbroken abscess (if possible) is inoculated freely, since the parasitic elements are scarce, on broth, glycerin agar, potato, carrot, etc., on all of which it grows well. Growth becomes visible in some days, and gradually increases. On agar, the colonies are first white and cream, and later a dirty grey. On carrot, the colour is first yellow, then grey, and finally quite black. On potato, small, white, woolly spots appear, increase in size, and change to a brownish colour. Further growth results in heaped-up masses likened to cerebral convolutions. " In gelatin stab, an inverted fir-tree growth is got, but no liquefaction. It forms acid specially with inulin in peptone solution, and also with glucose, maltose, galactose, raffinose, saccharose, and mannite ; but with lactose, dulcite, inosite, adonite, sorbite, and salicin, the medium remains alkaline. In no case was there any gas formation. No indol formation was observed. The organism was definitely aerobic " (Ritchie, loc. cit.). This observer has also studied the organism in hanging-drop agar cultures, and found that the mycelium formation is readily noted at 220 C, but at 370 C. few filaments are formed ; instead, large spores (5 micra) from which short stalks sprouted, each bearing a spore, which thus formed a circle round the central body. The latter soon degenerated. This suggests the reason 340 PUBLIC HEALTH BACTERIOLOGY why filaments are not usually found in the pus or granulo- mata. The optimum temperature therefore is about 20° C. (150 to 220). The organism is Gram-positive, but not acid-fast. Outside the human body, the organism has been found living on decaying vegetable matter. Sporotrichosis has also been described as occurring in dogs, rats, and in the horse. In the last it causes a lymphangitis with superficial granulomata of a benign nature, but important from having a resemblance to glanders, especially as it also at times prevails as an epizootic. Human infection from the horse has been reported in twelve instances in seven or eight years in North Dakota, U.S.A., where sporotrichosis in horses occurs moderately frequently. A case is also reported in a female bitten on both thumbs while holding a rat which had been inoculated with sporotrichosis. Serum agglutination and complement fixation have been found to occur in sporotrichosis, but are not specific, as the serum of patients suffering from other mycotic affections (thrush, actinomycosis) reacts to Sporotrichum Beurmanni, at least in the dilutions tried. Oidium albicans (sometimes called Saccharomyces albicans) is the cause of thrush (Gr. Soor ; Fr. Muguet), a localized disease of the mouth and pharynx, but also at times attacking the oesophagus, stomach, small intestine, caecum, and anus ; besides being occasionally found developing on the vulva, in the vagina, on the pre- puce, and the glans penis. On rare occasions it has been found in the bladder, the kidney, the lungs, the brain, and in the blood. The oidium is composed of cylindrical filaments, made up of joints 50 to 60 micra long by 3 to 5 micra in diameter. These give off branches, which bear spores by constriction at their free ends. Budding is also noted when grown in media containing sugars, allying it to the yeasts. Two varieties are described, one which liquefies gelatin and produces spores ; another which does not liquefy gelatin and yields small spores. It grows only in acid media. Like the yeasts, it can ferment sugars, but is not so powerful as they are. It is easily diagnosed microscopically. A fragment of the white membranous growth from the tongue or mouth YEASTS AND MOULDS 341 is taken on a slide, teased out in a drop of acetic acid (which renders the epithelial cells almost invisible), and examined. The parasite is clearly seen as described above. The spores are round or oval. A stained specimen may be made by teasing in a distilled water drop, exposing to dry, fixing by heat or absolute alcohol, and staining with thionin. To isolate in culture, inoculate on gelatin, and incubate at a low temperature (150 to 200 C.) for 48 hours. By that time white or creamy colonies appear, which are pure cultures of the thrush fungus. The ordinary microbes of the mouth are unable to develop at that temperature in the same time. Ringworm Fungi. — The study of these is very complex. The mode of demonstration of them in the various parts affected is the same for all, and is summarized thus by Agasse-Lafont : — Hairs. — Prepare a solution of caustic potash of 30 per cent strength. Extract some of the diseased hairs, and put them in a drop of this solution on a slide. Put on a cover-glass, and heat moderately for several seconds over the flame of a spirit lamp, until the hairs can be crushed by gentle pressure on the cover-glass. Examine directly without staining, with a dry lens and medium light ; or first mount in glycerin or glycerin jelly. Epidermal Scales. — Tease out with two sterile needles, and treat in the same manner. Nails. — Reduce to powder with a nail file, and treat as above. Pus. — Dry on slide, and examine directly without staining. Favus Crusts. — Tease out, crush between two slides, and thereafter treat as for hairs. They are best cultivated on Sabouraud's medium Agar, 18 grm. ; peptone, 10 grm. ; maltose, 40 grm ; and water to 1000 c.c. Heat to dissolve, fill into tubes, and sterilize on three successive days. To inoculate tubes, take an infected hair, rinse it for a few seconds in absolute alcohol, and wash thoroughly with sterile water. Then stab it into medium at several places, and grow at 180 C If first growth is not pure, remove plug, and inverting tube 342 PUBLIC HEALTH BACTERIOLOGY over another, tap it smartly, when spores of the fungus will fall into the other tube and inoculate it. At i8° C. growth appears in seven days as fine white downy tufts, which increase in size and throw out rays. The surface of the growth becomes covered with a fine white powdery material. If grown on gelatin, liquefaction takes place in twelve to fifteen days. The microsporon fungus gives a more delicate growth than the megalosporon fungus, and also shows microscopically club-shaped ends to some of the filaments, which are not found in the other. In both forms, spores are found on one side of the threads (like the teeth of a crab) or at ends like a bunch of grapes. Table of the Principal Ringworm Fungi (Agasse-Lafont). Trichophyton Trichophyton Microsporon Achorion tonsurans mentagrophytes audouini schoenleinii Patho- Ringworm of Tinea tonsur- Tinea ton- Favus of genic scalp : Tinea ans suppurates; surans, etc. scalp, skin, role tonsurans Body: Herpes circinatus Tinea barbce or Sycosis menti and nails Lesions Small : Small : Large : Sulphur- produced Hairs : broken close to the scalp, or short Suppurative : Hairs; bro- ken long, and having at their base a greyish sheath yellow crusts Principal i. Spores large 1. Spores 1. Spores 1. Mycelia of charac- w vary (2-10^) small (2~3M) varying thick- .ters of 2. In "^regular 2. In lines 2. In mosaics nesses fungus lines 3. Inside and 3. Spores 2. Wavy 3. Inside hair outside hair only on out- roots roots side of roots Trichophyton tonsurans. — This fungus is the cause of 30 per cent of the ringworm of the scalp, in Paris, and almost all the cases in Germany and Italy. In the east end of London, among the Polish, German, and Russian Jews, it is a common cause. It also is found in ringworm of the body, and at times causes an affection of the beard and the eyebrows, dry in character. Rarely, it affects the nails. YEASTS AND MOULDS 343 It is composed of simple filaments interlaced. In the hair bulbs, the filaments are inside the cuticle (endothrix) and- running parallel to the long axis, and are formed of cells or spores, almost square. These spores are 4 to 5 micra long, and are regularly arranged in lines. It is readily grown on Sabouraud's medium, showing in five to six days. It liquefies gelatin. A variety is quite frequently met with in the same places, T. Sabouraudi. It has a more fragile mycelium, and shows round spores. Trichophyton mentagrophytes is a cause of sycosis menti, a suppurative affection of the beard, and also of ringworm of the body, and a suppurating ringworm of the scalp in infants. The filaments are composed of strings of round spores of varying diameters (2 to 10 micra). The filamentary threads are mostly outside the hair cuticle ; (endo- and ectothrix) ; a few are found inside but towards the periphery. Microsporon or Microsporon Audouini is almost the sole cause of ringworm of the scalp in Scotland, and of 96 per cent of the cases in London among British subjects. It is called the small-spored fungus as compared with the two given above, which are called " megalosporon," or large-spored. The parasite encloses the diseased hair in a whitish case formed of a mosaic of spores (ectothrix) . The spores are 2 to 3 micra in diameter, and from pressure against one another in the mosaic pattern, become poly- hedral in shape. When stained with carbol-thionin, the filaments are seen in the interior of the hair. Achorion Schoenleinii is the fungus which causes f avus, or honeycomb ringworm, in which the characteristic feature is the formation of cup-shaped crusts of a sulphur- yellow colour. It most commonly attacks the scalp, but also affects the skin of the body and the nails. When attacking the nails, they become yellow and thickened. Besides the characteristic form of attack, the fungus may also produce a moist dermatitis resembling that due to the other ringworm fungi. In the hair the parasite is seen as wavy lines of mycelia composed of spores. The spores are irregular in size and shape, but mostly polyhedral. Rarely, the mycelium is septate and without spores. 344 PUBLIC HEALTH BACTERIOLOGY Favus prevails also in mice and cats, and from the latter animals many of the human cases arise. The Achorion Schoenleinii in culture grows best at 300 to 350 C, and scarcely grows at io° to 150 C, unlike the other ringworm fungi. It also liquefies gelatin more quickly than they, namely, in three to four days. It forms snowy-white circular or oval colonies, becoming finely powdered over the surface, and wrinkled in old culture. It grows best on beer-wort agar. To beer- wort diluted to a specific gravity of 1100 add 1-5 per cent of agar. Heat for two hours until dissolved. Filter, tube, and sterilize. (Avoid overheating.) CHAPTER XV III. SPECIAL BACTERIOLOGICAL EXAMINATIONS. BACTERIOLOGICAL EXAMINATION OF WATER. In all natural unfiltered waters, except when derived from deep wells and springs (in which case filtration has already taken place through the strata) numbers of bacteria are found. The actual content is determined by the accumulated action of the following factors, namely : — i. Presence or absence of local pollution. 2. Presence or absence of natural purification. 3. The season of the year. 4. The rainfall at any particular period. The bacteria found in water may likewise be classed under four heads, as — 1. Harmless : the natural water bacteria. Such are the B. fluorescens liquefaciens, B. fluorescens non-liquefaciens, B. prodigiosus, B. violaceus, sarcinae, and spirilla. These all grow best at room temperature. 2. Unobjectionable : those present from soil washings, as B. subtilis, B. mycoides, and B. megatherium. 3. Objectionable : those derived from sewage, either directly or from sewage-polluted soil. Such are : (a) The B. proteus group ; (b) B. coli communis and its allies ; (c) Streptococci ; (d) Staphylococci ; (e) B. enteritidis sporogenes. 4. Dangerous : those capable of causing infection by the alimentary canal. Such are the B. typhosus, B. para- typhosus, B. dysenteriae, and Sp. cholerae. Samples. — Stoppered sterile glass bottles should be used for sampling, each of at least 250 ex. capacity (8 to 10 oz.). The bottle should be thoroughly cleansed with soap and water, well rinsed with clean water, and sterilized (inverted and with stopper out) in steamer for one hour. 346 PUBLIC HEALTH BACTERIOLOGY Allow the sterilizer to cool, then stopper the bottle, and remove and put in case. In sampling from a tap, run the water to waste for half an hour, and then fill bottle. Stopper, and label with particulars of place, time, and date. Examine at once, or pack in ice to prevent multiplication of organisms. In sampling from a lake, dip stoppered bottle well below surface ; remove stopper, and keep it under water ; allow bottle to fill ; replace stopper, and bring bottle to surface. Pack in ice. Examine samples as soon as possible after collection. Keep in ice in meantime. Dilutions. — If the water is pure, no dilution will be required ; if impure, varying dilutions are used according to the degree of impurity. These may be made by the decimal mode of dilution described on page 367 ; or flasks may be kept ready containing 100 c.c. of sterile water. One c.c. of sample added to such a flask by sterile pipette gives practically a dilution of 1 in 100 ; 1 c.c. from this flask to another sterile 100 c.c. gives 1 in 10,000 ; and so on. To get 1 in 10, remove 10 c.c. by sterile pipette, and add 10 c.c. of sample ; and from this dilution others are simi- larly made. Standards. Deep wells and springs \ Surface waters : — Shallow wells Cultivated lands Rivers Bacterial Count. Gelatin Plate at 200 C. Should not exceed 50 per c.c. Ditto 500 per c.c. Agar Plate at 37° C. Should not exceed 10 per c.c. Ditto 50 per c.c. Bacillus Coli Communis. Should be absent in 100 c.c. Ditto in 10 c.c. Methods of Water Examination. — These are based on the knowledge that the dangerous organisms in water are usually present from sewage pollution. Inasmuch as some of these forms are not easily isolated from water, the mode of procedure is to ^enumerate the total bacterial content, and to look for an organism, likewise present from SPECIAL EXAMINATIONS 347 sewage pollution, but easily found if present. Such an organism is Bacillus coli communis, which is present in enormous numbers in the sewage of man and animals ; and is therefore likely to be present in sewage contamin- ated water, even after great dilution. The B. coli is also a more resistant organism than the dangerous forms, and so serves to indicate pollution at a later stage than these could possibly be found in a water. So far it is not possible to distinguish B. coli of human origin from those of animal origin. It is stated that those of human origin are more pathogenic to animals. There are various methods in use in this country. A Committee of the Royal Institute of Public Health appointed to consider the " Bacterioscopic Examination of Water," reported in 1904 {Journal of State Medicine, vol. xii, p. 471) as follows : — Minimal Procedure. Unanimous report : — (a.) Enumeration of bacteria present in a water sample, capable of growing on a medium incubated at room temperature (i8°-22° C). (b.) Search for Bacillus coli, and identification and enumeration of this organism, if present. Majority also recommended : — (c.) Enumeration of bacteria present in sample capable of growing on a medium incubated at blood heat (360-38° C). (d.) Search for and enumeration of streptococci. (e.) Do not recommend routine examination for Bacillus enteritidis sporogenes ; but in exceptional and special cases advise that it be searched for. They further report on mode of collection of sample, media to be used in the tests, etc., of which the following is a brief summary : — Collection of Sample. — The sample should be collected in sterile stoppered glass bottles (minimum quantity 60 c.c, or 2 ounces), and should be packed in ice. At least 10 ounces of sample should be sent, and its examination should be begun within three hours, or it should be left packed in ice. Enumeration of Bacteria. — The media to be used are all to be standardized to have a reaction of +10 (Eyre's scale). Owing to changes in reaction, media should not be more than 348 PUBLIC HEALTH BACTERIOLOGY three weeks old. For cultivation at room temperature a choice may be made from the following : Distilled-water gelatin, nutrient gelatin, distilled-water agar, nutrient agar, and gelatin agar. At blood heat use agar or gelatin agar. Both agar and gelatin media should be used in any one test. Polluted water gives more colonies on nutrient gelatin than on distilled-water gelatin ; unpolluted water gives more with distilled-water gelatin. The size of the plates, the amount of medium to be used in plating, and the amounts of sample to be added to the media are all specified. Plates should be 10 cm. in diameter ; 10 c.c. of medium should be used ; and for ordinary waters, o«2 c.c, 0-3 c.c, and 0-5 c.c. of sample should be added to gelatin media, and o-i c.c. and i*o c.c of sample to agar media. The sample should be thoroughly shaken before removing these amounts, and the tubes should be thoroughly mixed by rota- tion before plating. Duplicates should be put up. In an unknown water, additional plates of dilutions (ten and one hundred fold) should be made. The colonies should be counted by the naked eye, and preferably by daylight. A lens may be used for doubtful colonies. The time of counting should be : For gelatin plates, at the end of 72 hours (3 days) ; For agar plates, after 40 to 48 hours. The gelatin plates should be inspected daily, in case counting becomes necessary earlier from liquefaction of the medium. Search for B. Coli. — MacConkey's broth is recommended to be used, the sample to be added directly to the medium, and not first concentrated by filtration. Isolation of B. Coli. — If indications of the presence of B. coli are got, then the organism must be isolated, cultivated, and identified. This is advised to be done by making surface cultures on plates of either : — (a.) Litmus lactose agar (reaction +10) ; or (b.) Bile-salt lactose agar (MacConkey's) ; or (c.) Nutrose lactose agar (Drigalski and Conradi) ; or (d.) Ordinary nutrient gelatin. They consider (c) to be the best medium of all. Agar media save time. Identification and Tests. — Having obtained coli-like colonies on plates made from the preliminary cultivations of the water in MacConkey's broth, subcultures must be made to identify the organism. The following subcultures should at least be made : — (a.) Surface Agar, on which the abundant growth enables subcultures, etc., to be easily made if required. SPECIAL EXAMINATIONS 349 {b.) Stab and surface cultures in gelatin. These may- be done in the same tube. (c.) Litmus milk incubated at 37 ° C. (d.) Glucose litmus medium. (e.) Lactose litmus medium. (/.) Peptone water for indol reaction. The Committee consider an organism to be typical B. coli when it conforms to the following characteristics : Small motile bacillus, non-sporing, decolorized by Gram, which grows well at 37 ° C. and at room temperature ; never liquefies gelatin ; produces permanent acidity in milk ; curdles milk within seven days at 370 C. ; ferments glucose and lactose, with formation of acid and gas ; grows in smooth thin surface on gelatin (not corrugated) ; and in gelatin stab grows well to the bottom of stab. This typical B. coli generally also forms indol, gives a thick yellowish- brown growth on potato, changes neutral-red, reduces nitrates to nitrites, and in fer- menting glucose half of the gas produced is absorbed by KOH. It sometimes ferments saccharose. The method here described is that used in the City Bacteriological Laboratory, Glasgow. I. Enumeration of Bacteria. — Add o-i c.c. and 1 c.c. of sample by sterile pipette to 10 c.c. of liquefied gelatin and agar. Mix thoroughly by rotation and plate. Incubate the gelatin plates at 20 ° C. ; inspect daily, and count after three days, unless necessary earlier. Incubate the agar plates at 370 C, and count after two days. The ordinary water bacteria grow best on gelatin, whereas the intestinal forms grow best on the agar at blood heat. Hence, in a pure water the gelatin count should be much the greater ; and in an impure water the difference between the counts becomes less marked the more impure the water. The ratio between the two counts is also noted. This ratio of the number of organisms developing at room temperature to the number at blood- heat = 10 : 1 in pure water and = 10 : 2 or 3 or 5, etc., in polluted waters. The ratio is unreliable in surface waters in tropical countries, because B. liquefaciens, B. fluorescens liquefaciens, and B. fluorescens non-liquefaciens are com- monly present, and all grow well at 37°C., and are harmless. 350 PUBLIC HEALTH BACTERIOLOGY Average Number of Bacteria per Growing on Gelatin c.c. of Water Sample AT 20° C. Glasgow Tap Water. Raw Thames Water. Year 1909 : — Highest Lowest Average j 73 (Dec.).. 31 (June).. 54*2 19,794 (Dec.) river in flood. 913 (May) river low. 3,818. Year 1910 : — Highest Lowest Average 105 (Jan.) . . 19-5 (May) . . 43-o 22,939 (Dec.) river in flood. 1,522 (May) river low 7>4i°- Dr. A. C. Houston (Director of Water Examination to the Metropolitan Water Board, London), in addition to gelatin and agar media, for enumeration, also uses MacConkey's neutral-ral, fo'le-salt, peptone, lactose 3 gar (called rebipelagar for brevity). This is similarly inocu- lated and plated and incubated at 37 °. The colonies are counted on gelatin on the third day, and on agar and rebi- pelagar after twenty to twenty-four hours. The following table, culled from Dr. Houston's reports, serves to illustrate the ratios of the various counts : — RAW RIVER THAMES WATER. Average Number of Microbes per c.c. in Comparable Samples, Tested on Three Media ; with Ratios. Gelatin. Agar. Rebipelagar. Ratio. Ratio. Year. Gelatin. Agar. Agar. Rebipelagar. 1908-09 2745 319 38 8: I 8: 1 I909-IO 531° 495 63 11 : 1 8: 1 1910-II 6184 339 20 18: 1 17: 1 Gelatin at 2o°-22° C. ; colonies counted on third day. Agar and Rebipelagar at 370 C. ; counted after twenty to twenty- four hours. This further table (curtailed) from Houston's Fifth SPECIAL EXAMINATIONS 351 Annual Report (page 7) shows the influence of rainfall in the Thames valley on the average daily flow of the River Thames, and on the bacterial content of the raw river water : — Average daily (natural) flow of the River Thames in million gallons. Total number Percentage number of samples of raw Thames Water containing typical B. Coli in o*i c.c, Raw Thames Month. Rainfall (inches) Thames Valley. of bacteria per cc. in the raw Thames Water (gelatin) Water. Oxygen absorbed from permanganate test (parts per 100,000) 1910 : April 2-13 1353 3IO9 23'7 •1352 May I'96 979 1522 12-5 •I489 June 3-04 1149 2721 500 •3031 July 2-25 795 2589 526 •1756 August 2-88 589 2702 13*7 •1357 September 0*46 501 3035 13-6 •1173 October . . 3-48 666 3736 38-1 •1611 November 361 1595 17932 682 •3249 December 5-21 5064 22939 83-3 •5253 1911 : January . . 1*21 2657 10438 857 •1852 February 1-67 1443 8035 700 •1140 March 1-99 2033 9300 78-2 •2495 Sum 3°'49 Averages 2-54 1574 7324 49-4 •2154 Note. — The figures in bold type exceed their respective averages. II. Search for B. Coli. — For this purpose MacConkey's broth is used of the composition neutral-red, bile-salt, peptone, glucose water,* in single, double, triple and quad- ruple strengths. The proper quantities are put into suitable sized tubes, and fermentation or Durham's tubes * Just as neutral-red, bile-salt peptone, lactose, agar has been shortened to rebipelagar (see p. 350), so MacConkey's broth with the various carbohydrates might be written thus : — ■ Neutral-red, bile-salt, peptone, glucose water (aqua) as rebipegluqua. lactose „ „ rebipelaqua. saccharose „ „ rebipesaqua. dulcite „ „ rebipeduqua mannite , „ rebipemaqua. adonite , „ rebipeadqua. inulin , , „ rebipeinqua. 352 PUBLIC HEALTH BACTERIOLOGY added, and the whole sterilized. Thereafter, the sample, having been well shaken, is added direct by sterile pipette, and always without concentration by filtration. If the sample is too strong, suitable dilutions are made, and i c.c. of the dilution is added. In the case of an unknown water the following tubes would be put up : — o-oooi c.c. of sample to 10 c.c. of single strength medium. o-ooi J J J) o-oi >> ) o-i >> y i-o ,, , io-o >> > 50*0 >> > ioo-o ) > > of double strength. 25 c.c. of triple ,, 30 c.c. of quadruple ,, The tubes are put in a nest or basket, and incubated at 370 C. for twenty-four hours. The possible results are four : — (a.) Acid and gas. (b.) Acid, no gas. (c.) No acid, no gas ; turbidity. (d.) No visible change. Interest lies in (a) and (b), and they are commonly associated ; but the absence of (a) from all the tubes should not be held as precluding the necessity for further investigation. Note the tubes showing acid and gas, or acid alone ; say that the tube containing least amount of sample, and which shows acid and gas, is that to which o- 1 c.c. of sample was added, then the result is stated thus : Sample showed acid and gas formation down to o-i c.c. As a rule all the higher tubes will show acid and gas too. Some workers call this the " presumptive B. coli test ; '* but as the following paragraphs (from Notter and Firth) show, the test is only a step on the way towards the isolation of B. coli. MacConkey's Neutral-red, Bile-salt, Peptone, Glucose Water. Reaction of certain bacteria with : Group i. Bacteria producing acid -f- gas. B. coli communis, B. enteritidis (Gaertrier), B. paracolon, B. paratyphosus, B. pneumoniae, B. lactis SPECIAL EXAMINATIONS 353 aerogenes, B. acidi lactici, B. neapolitanus, B. icter- oides, B. psittacosis, B. cloacae, B. proteus vulgaris, bacillus of hog cholera, bacillus of epidemic jaundice, B. oxytocus perniciosus, and B. capsulatus. Group 2. Bacteria producing acid, but not gas. B. typhosus, B. dysenteriae, B. cholerae, B. pyo- genes fcetidus, streptococci and staphylococci. The first group can be subdivided into four : — (a.) The proteus group of motile bacilli : gelatin-lique- fying, form acid and gas in glucose, maltose, saccharose, and galactose, but not in lactose, laevulose, arabinose, rafhnose, mannite, sorbite, dulcite, adonite, dextrin, starch, or inulin ; curdle milk slowly with acid ; commonly produce indol in peptone solutions. (b.) B. coli communis family : motile bacilli, non-gelatin- liquefying, producing acid and gas in all the above except saccharose, adonite, starch, or inulin ; curdle milk rapidly with acid, but do not peptonize clot ; form indol. (c.) B. lactis aerogenes group : non-motile bacilli, non- gelatin-liquefying, producing acid and gas in all the above except three — dulcite, adonite, and inulin ; curdle milk rapidly with acid ; do not peptonize clot ; form indol. (d.) The paracolon-enteritidis series : motile bacilli, non- gelatin-liquefying, producing acid and gas in all but lactose, saccharose, adonite, starch, or inulin ; do not clot milk but finally render it alkaline ; do not form indol. III. Isolation of B. Coli.— (a.) From each tube showing acid and gas, and acid alone, make a surface smear with one platinum loopful on a plate of neutral red, bile salt, peptone, lactose, agar (rebi- pelagar) containing 1 part of crystal violet in 10,000. Incubate these plates for twenty-four hours at 370 C. (b.) Examine thereafter. If a plate shows only one kind of colony, inoculate an agar slope from a mixture of these. If more than one form of colony is seen on any plate, inoculate agar slopes from each kind of colony. Incubate the various inoculated agar tubes (properly marked or labelled) at 370 C. for twenty-four hours. This growth on agar gives sufficient material for subsequent steps ; but it is also necessary to revive any fermentative powers 23 354 PUBLIC HEALTH BACTERIOLOGY •aiiqioS •unnui +++ :+ +++ +++ + + + + + I I II I + I ++ + + + l I I I l *3}TUOpV + + + + I ++ I III ++ I I Mil •ajiiroej^ •3}p[n £ ft 1 + 1 1 1 1 1 1 1 1 1 1 l 1 1 : l l l d CO ■ bo OJ a i 9 CO CU tlD +++++++++++ 1 < 1 < i <<< 1 2 -d <++<<+<++++ 1 l 1 i \< 1 1 q o3 a CU 4> > II 1 1 1 II 1 1 1 1 1 l 1 1 \< t 1 12 'o Oj CO § ho CO d 1 1 1 1 1 1 1 1 1 1 1 1 l 1 1 KM O m" CO '3 en M co ft +.++++++++++ + < << I <<«: 1 C o o > PQ M M MM aaa C pp OT I I 1 I l l l l l I l 1 1 1 1 i i i i o d ■5 CU d W) +++++++++++ + + 1 1 + i|i 1 S S o o I o3 OQ ~ g d 1 O u & ~u~ fcuB a •2 3 a.) : k.) . uelle Kay uelle 0 o o C 2 | pq ides (Sanarelii) . . (Hume) (MacConkey) g cholera (Smith, M „ (Smith, Ar tifer Dion (Day) (Le Sage) . . pphosus A (Schottm „ A (Brion & B (Schottm "5* &a '3 2 enes dus losis rodent 3 •ST .9 3 s 5 1 o a oj oj o o d d o o M O PQ co" d co " o o d co O d U CO Group : teriae (SI (F s alkalig nes foeti otubercu | o 8 d 03 o o d 3 •5 "5 o o U U ft ft .2 8.1 O ; ; O £ o a** 5" si. b/> . O +-> 4> . . ers : faecali pyoge pseud pestis CO | 5 S 5 3 u i tfqpqpQpqpqpqpqpqpqpqpq S« £« ^pqcq^Mpqpqpq co ^J O g h Q ( 0 + 1 , 3,8i6 „ 5 Very slight cream, £" deposit 5 » 2,196 „ 6 „ „ |* „ . . 4 M 636 „ 7 » „ i" „ • ■ 2 „ 1,104 „ 8 Very watery. Thin layer of cream, i" pus-like deposit 0 ,, 396 „ Cow No. III. — i Watery, slight cream, \" pus- like deposit o 168 bac. 3 Ditto ditto \" ditto o 192 4 Ditto ditto ¥ ditto o 252 „ (Milk from quarter only amounted to about 2 ozs.) Cow No. IV. — i Watery, slight cream, £" deposit 120 bac. 5,172 bac. r ,. r ., very slight cream, |" deposit heavy cream, -^" deposit ¥ blood- stained deposit 1,020 1,212 1,584 364 2,532 1,824 6,804 5,9io 14,460 10,908 5.316 4.824 From the samples taken from Cows II., III., and IV., it was clearly shown that the deposit is the proper part of the specimen to take for the examination, it containing by far the greater number of tubercle bacilli. 4. For Actinomyces. Make a film from sediment, and stain by acid-fast method. Actinomycosis in the udder of the cow is usually alleged to be rare, but is stated to be more common in the sow. In an article on " The Occurrence of Actinomycosis in Cows' Udders," by Dr. J. Hume Patterson, in the Journal of Meat and Milk Hygiene (vol. i, SPECIAL EXAMINATIONS 371 No. i, Jan. 191 1), the writer cites evidence which goes to show that it may not be so uncommon. Out of fifty specimens from different udders submitted to him for suspected tubercle, in five cases the lesions proved to be actinomycotic. The lesions in each case were indistinguish- able by the naked eye from those of tuberculosis. On cutting into the substance of the udder numerous cream- coloured foci, similar to tubercles, were seen, ranging from the size of a pin-head to that of a pea. The part affected was also of a brownish tint, as is so often seen in tubercu- losis of this tissue. Smear preparations showed elements of actinomyces in four of the cases ; in one, no elements were found, but on making sections typical actinomyces were found. All the others were confirmed by making paraffin sections. In one of the smears both tubercle bacilli and actinomyces were found ; and if not on one's guard, such a case could be readily dismissed as tuber- culosis without looking for actinomycosis. Dr. Patterson says : "I am confident from my experience in these cases that if each suspected tubercular udder were subjected to a microscopical examination, the percentage of actinomy- cotic udders would be greater than is generally supposed. In regard to milk, many of the samples, taken by veteri- nary surgeons inspecting dairy herds from cows having what appeared to be marked tubercular lesion of the udder, have proved negative even on animal inoculation. Dr. Patterson asks the question : " Might these not be cases of actinomycosis ? " and cites the following case : — " During last winter's inspection (1909-10) a case occurred where the lesion of the udder was markedly nodular and similar to tubercle. A sample of the milk was taken, and a guinea-pig inoculated with negative result. Not satisfied with this result, samples were again taken from all four quarters of the same cow. These samples again proved negative on animal inoculation. Smear preparations from these last samples, made from the deposit of the centrifugalized milk, showed a few acid-fast rod-shaped, and a few fragments of club-shaped, elements suggestive of actinomyces, in those samples taken from both hind quarters and from the left fore quarter. I was unable to procure the udder for further examination, but am convinced that this was a case of actinomycosis. In actinomycosis of the human subject, it is 372 PUBLIC HEALTH BACTERIOLOGY yet doubtful how infection takes place, as the cereal theory- has been partly exploded, through cases arising which had no connection with grain; and I think it is just possible infection may be conveyed by the milk of such a cow as I have quoted, where the elements of the disease were found in the fluid. If that be so, this disease, as affecting the udder of the cow, warrants more attention than is given to it at the present moment in connection with our milk supplies." In this regard the present writer was consulted in 19 10 by a young man of about twenty years, who had recently returned to Scotland from Canada. He had been treated in several hospitals for tuberculosis. The history being irregular, his spit was sent to the City Bacteriological Laboratory (Glasgow) for examination with this note : "If you do not find tubercle bacilli, look for something else." Dr. Sutherland, who examined the speci- men, found actinomyces, and the diagnosis was confirmed later in the Glasgow Royal Infirmary. He died some months later, but a post-mortem examination was not obtained. The history bearing on the point at issue is this : About two years previously he left his position as an office boy in Glasgow, and went to near Calcary, Alberta, Canada, where he became a farmer's boy. One of the cows he had to milk had a chronic sore on its udder, and he was warned to milk this cow gently, so as not to make the sore bleed. After about one year he took ill at this farm with a sore throat, followed by a swollen right submaxillary gland. The gland was incised and healed well. Then another swelling appeared, and another, and so on. This certainly looked like a milk infection, but verification was not possible. 5. For Johne's Bacilli. The prevalence of Johne's disease in cattle in Great Britain being now well established, the bacilli may find their way into the milk from the diarrhceal stools in the earlier stages of the disease. The bacilli are shorter than the tubercle bacilli, but are equally acid-fast and alcohol- fast. Twort has cultivated them on egg media. 6. For Streptococci. Houston advises the use of the medium of Drigalski and Conradi (lactose-nutrose-agar) in plates. The plates are inoculated by smearing over the surface of them o-i c.c. of each of the dilutions given above. Incubate twenty-four to SPECIAL EXAMINATIONS 373 forty-eight hours at 37 ° C, and subculture the minute colonies formed into broth, and repeat cultivation as to time and tem- perature. Make films from the broth and examine micro- scopically, and if found in pure culture, subject the organism to the differential tests for streptococci and to nitrate broth test. According to Houston, 58 per cent of the Streptococci faecalis of the cow are of the lamirasacsal variety ; that is, clot milk and ferment lactose, rafhnose, saccharose, and salicin. It forms short chains, and is not pathogenic to mice. The S. pyogenes does not clot milk, ferments lactose, saccharose, and sometimes salicin, but does not ferment rafhnose, and is pathogenic to mice, forms long chains, and does not reduce nitrates. Leucocyte Test. — In testing for chronic mastitis in cows, Trommsdorff found that the deposit of leucocytes after centrifugalizing the milk in a specially-shaped tube, was a good guide as to the necessity for further investigation. In an enquiry on these lines, he found 20 per cent of chronic mastitis in cows, and it was associated with the presence in very large numbers of capsulated streptococci. Such cows give less milk The milk must be drawn directly from the animal before one can say that the streptococcus is from the udder of the cow. Centrifugauzation of Milk Is used to precipitate the gross dirt, pus cells and leucocytes, and bacteria. According to Scheurlen, the ordinary milk bacteria in the proportion of 75 per cent of them go into the cream, as do also the organisms of anthrax, typhoid, and cholera. The other 25 per cent of these remain in the separated milk. On the other hand, tubercle bacilli are largely found in the sediment, a few only passing into the cream and separated milk. Fermentations. Lactic Acid. — The development of acid and curd occurs normally in milk on keeping It is due to the formation of lactic acid from the milk sugar or lactose, by the action of enzymes produced by microbic growth. Many species of bacteria are able to produce the lactic fermentation, but in milk those most commonly causing it are : (1) Bacillus 374 PUBLIC HEALTH BACTERIOLOGY lactis aerogenes, and (2) Streptococcus lacticus (Kruse), identical with the B . a cidi lactici (Leichmann) . Heinemann, who has investigated the subject, states that the two species are ordinarily present in naturally souring milk, the former in abundance at the beginning, the latter in the later stages when the acidity has reached a high degree. The secret of the regularity of the presence of these two species is their power of withstanding a much higher degree of acidity than the other species present at the first. In changing to lactic acid, the lactose is believed to be first hydrolyzed into glucose and galactose. CifH.iOu + H20 = C6H1206 + C6H1206 = 4C3H603 Coagulation of casein follows on acidification of the milk, the amount of acid necessary to precipitate the casein averaging 0-45 per cent ; the terminal amount may reach 0-85 per cent. The casein precipitated by lactic acid formation is never redissolved, because the high acidity inhibits the proteolytic ferments. Casein precipitation, however, may also be due to a non- acid coagulation caused by bacterial ferments. Casein precipitated in this way may be redissolved by a bacterial trypsin or casease, produced by the same or other bacteria, and the milk hence may become entirely liquid, transparent, and of a yellowish colour. B. bulgaricus in milk culture produces 2-5 per cent of lactic acid, and 0-05 per cent of acetic and succinic acids, is non-pathogenic, and exerts no putrefactive action upon proteids. Metchnikoff suggested its use in milk cultures as a food to inhibit, by its acid production, the growth in the intestine of the class of bacteria which break up proteids, the bacteria of putrefaction. This is Metchnikoff 's bacteriotherapy, which has been extensively practised. B. bulgaricus is a large, non-motile, non-sporing, Gram- positive bacillus, with square ends (like B. anthracis). It forms short and long chains. It shows little or no growth on ordinary media or below 370 C. Optimum temperature : 42 ° C. Grows in dextrose-peptone broth, to which calcium carbonate has been added. Butyric Acid fermentation of milk occurs occasionally in milk from the growth of anaerobic bacteria. It is a SPECIAL EXAMINATIONS 375 much slower process than the lactic one, and can also be produced by some pathogenic anaerobes, e.g., bacilli of quarter-evil, malignant oedema, B. Welchii and B. enteritidis sporogenes (Klein). Alcoholic Fermentation of milk occurs spontaneously on rare occasions. The process is due to the natural in- troduction of yeasts, and once started can be kept going by infecting fresh milk. Koumiss is thus made by the Tartars from mare's milk. Kefir is an effervescent sour milk made from cow's milk by the addition of " kefir grains," little cauliflower-like excrescences whose fermen- tative power is due to Saccharomyces mycoderma. Mare's milk is more suitable for the preparation, because the lactic fermentation also accompanies the other, and the duration of the double fermentation is conditioned by the amount of sugar, which is 5-5 per cent in mare's milk and 4-8 per cent in cow's. The latter is richer in casein and fat, and both of these constitute disadvantages, so that it is usually diluted in making koumiss. In the making, the milk is stirred constantly by day but rested at night. The amount of alcohol in all these products — koumiss, kefir, and cow's milk koumiss — is under 2 per cent, and the Tartar is capable of consuming three to four gallons of such milk on a hot summer's day without becoming more than hilarious, and with no digestive disturbance. They have been much used as " consumption cures." Diseases of Milk. Unusual or abnormal changes in milk are sometimes referred to as " diseases." They are produced by bacteria which have got into milk in various ways. Blue, green, and yellow milks are due respectively to the bacilli cyano- genes, erythrogenes, and synxanthus ; and red milk to B. prodigiosus. Bitter milk is due to a number of species, yeasts and diplococci having been isolated. Slimy or ropy milk has been traced to B. lactis viscosus, said to be a water organism. Slimy milk is produced at Edam (Holland) by the use of a streptococcus, for the manufacture of Edam cheese. Soapy milk is due to a micrococcus derived from fodder. 376 PUBLIC HEALTH BACTERIOLOGY BUTTER. Butter is made from the cream of milk by churning or agitation, whereby the globules of fat are broken, or rather have their casein envelopes ruptured, and then the fat globules adhere. The cream is first allowed to sour, otherwise the butter will be flavourless. The souring is) brought about by bacteria, nearly all of which are lactic acid formers. Tuberculosis is the only infective disease transmitted by butter. Tubercle bacilli have been found alive and virulent in butter after having been kept in a refrigerator for five months. Rabinowitch's acid-fast butter bacillus is easily distinguished culturally from the tubercle bacillus. Foot and mouth disease has been reported as having been transmitted by butter. Typhoid infection is unlikely, and has not yet been definitely traced. CHEESE. Cheese is the precipitated casein of milk, the casein or curd being insoluble. In hard cheeses the whey is better expressed than in soft cheeses, and so the sub- sequent ' ripening " which is due to bacterial growth is less in the former than in the latter. In the ripening of the curd, three groups of bacteria are engaged, (i) acid pro- ducers, like B. acidi lactici, (2) casein digesters, which break down the curd, and (3) gas producers, which honeycomb it. The actual organisms engaged have been determined in the case of particular cheeses, and are : a bacillus resembling the Bacillus subtilis, a mould (Oidium lactis), a penicillium mould, and yeasts. Tubercle bacilli of the bovine and human types have been found in cheeses. SHELLFISH. For Houston's methods, see Journal of Hygiene, vol. iv, No. 2, p. 185. WATERCRESS AND OTHER VEGETABLES. See Report to the L.C.C., by Houston, 1905. SPECIAL EXAMINATIONS 377 DISINFECTANTS. A disinfectant or germicide is a substance which destroys the microbic causes of disease. The same substance in a weaker strength may act as an antiseptic, that is an agent which restrains or checks the growth of bacteria without destroying them. A deodorant is a substance which destroys or masks the offensive effluvia or vapours resulting from bacterial growth, and may or may not have an action on the bacteria themselves. The best example of an agent capable of all these functions is potassium permanganate, which in strengths of 5 per cent and over is a disinfectant, under 5 per cent is an anti- septic, and in all strengths is a deodorant. (Solubility, 1-18 of water ; 1-3 boiling.) The mode of action of disinfectants varies. Some, like the strong acids, char or carbonize the bacterial body by oxidizing the other elements present. Others coagulate the bacterial proto- plasm ; while some, diffusing through the cell wall, exert a poisonous action on the protoplasm. Antiseptics like sugar, probably act by osmotic pressure through the cell wall, causing a flow of water out of the cell, thus drying up the protoplasm and rendering the bacterium inert for the time being. The chief disinfectants are the metallic salts, acids bases, the halogen elements, oxidizing agents, alcohols phenols, aldehydes, and the essential oils. The salts, acids, and bases act best in watery solution as against alcoholic solutions. Two explanations of this are offered : (1) That the salts, etc., in water dissociate into their ions, and the latter are more active in producing chemical change ; in alcohols, dissociation does not take place. (2) That the alcohol hinders the action of the disinfectant by hardening the bacterial cell wall. In favour of the second reason it has been noted that while absolute alcohol is useless as a germicide, added to aqueous mercuric chloride solution in the proportion of 25 per cent it increases the efficiency of the disinfectant. The addition of NaCl, on the other hand, diminishes the efficiency, it is believed by reducing the number of free ions. The halogens (CI, Br, I) are efficient in the order given. Chloride of lime liberates free CI when treated with an 378 PUBLIC HEALTH BACTERIOLOGY acid. When used simply in solution, oxygen is liberated in the nascent state, and oxidizes any organic matter present (CaOCl2 + H20 = CaO + 2HCI + O). Peroxide of hydrogen and potassium permanganate act similarly as oxidizers. Carbolic acid (phenol) and the cresols (lysol and creolin) do not dissociate, and their efficiency is increased by the addition of NaCl and diminished by alcohol. Formalde- hyde is not helped by adding salt, but alcohol is harmful. Standardization. — The emciency of any disinfectant depends on many factors, namely, the strength used, the solvent, the temperature, the bacterium to which applied, the time allowed for action, the other substances present, the number of bacteria present. For practical purposes the strengths are expressed as percentages ; but in com- parisons it is more scientific to work with solutions of the molecular weight or multiples thereof in one litre. Coefficient of Inhibition. — This term is applied to the strength (of a chemical substance) which is able to prevent the growth of a micro-organism ; that is, its antiseptic value. It is determined by making broths or other media containing the chemical substance to be tested, in a range of strengths. Equal quantities of the bacterium used in the test are inoculated into the various tubes (say, one loopful), the contents mixed, and incubated. In the case of solid media, they are poured into plates and the colonies (if any) counted. In broth cultures, look for turbidity, and confirm positive or negative results by making films. The coefficient is expressed in terms of strength and bacterium used. Thus, carbolic acid is said to inhibit the growth of anthrax bacilli when present in a strength of 1 part in 800 of medium. B. typhosus requires 1-400, and Sp. cholerae, 1-600. Of corrosive sublimate, 1-100,000 inhibits B. anthracis, and 1-60,000 inhibits B. typhosus. Germicidal, Bactericidal, or Disinfectant Strengths. — Koch used anthrax spores dried on silk threads, which he immersed in various strengths of substance being tested, at a definite temperature and for varying times. SPECIAL EXAMINATIONS 379 The threads were then removed and carefully washed in sterile water to discharge the disinfectant. They were then laid on gelatin and incubated, and the result was noted. This method is faulty, in that it is difficult to remove the disinfectant, some of which clinging to the bacteria inhibits growth. The Rideal-Walker method may be applied here to determine germicidal strength without comparison with carbolic acid. The results are stated in terms of strength used, time exposed, and bacterium killed : thus carbolic acid is bactericidal to anthrax spores at ordinary temperatures in 1-20 dilution in four to forty-five days ; at 40°C, in three hours. To Sp. cholerae at ordinary temperatures, 1-200 is fatal in five minutes, and 1-300 is fatal in two to twenty-four hours. To B. typhosus at ordinary temperatures, 1-50 is fatal in five minutes. To staphylococci and streptococci at ordinary tempera- tures, 1-60 is fatal in five minutes. Corrosive sublimate is fatal to anthrax spores in 1-2000 in twenty-six hours. To anthrax and typhoid bacilli and Sp. cholerae, in 1-2000 in five minutes. To staphylococci and streptoccoci, 1-10,000 to 1-2000 in five minutes. These tests supply useful data, but cannot be taken as applying to the action of the same disinfectants, when mixed with the body fluids. Rideal-Walker Test. — In this test carbolic acid is taken as the standard disinfectant, and the results of tests of other disinfectants are expressed in terms of their power, compared to the standard, of inhibiting growth of the same organism in the same time. This is called the " carbolic acid coefficient " of the particular disinfectant. Process.— A series of accurate dilutions of pure carbolic acid and of the disinfectant are prepared in sterile distilled water. A twenty-four hours' culture at 370 C. in Lemco broth (reaction + i*5) of B. typhosus is used as the test organism. A standard size of platinum loop is used to make the subcultures. The disinfectant solutions are 380 PUBLIC HEALTH BACTERIOLOGY arranged in a series of tubes, each containing 5 ex., and the dilution of the disinfectant is marked on each tube. To 5 c.c. of a particular dilution, five drops of the filtered culture are added ; the tube is shaken and set down. Now repeat with the next dilution, and so on. At the end of 2 -5 minutes, a subculture is made from the tube first inoculated, into 5 c.c. of sterile broth, and similarly with all the dilutions, in the proper order. At the end of 5, 7-5 10, 12-5, and 15 minutes, put up further subcultures thus, making six series of subcultures in all from each tube. The same process is carried out, at the same time, with the same culture, with dilutions of carbolic acid. All the sub- cultures are incubated for at least forty-eight hours at 370 C, and the presence or absence of growth is noted. From the table of results, the two dilutions doing the same work in the same time are seen. Say that 1-500 of Disin- fectant A inhibited growth in 2-5, 5, 7-5 minutes' exposures, and that carbolic acid 1-110 did the same ; then the carbolic acid coefficient of Disinfectant A is 500 -=- no — 4-5. Since its introduction this test has been much used. It has also been subjected to much criticism. It deals with " naked " bacteria. The other objections are that it must be carefully done, all the dilutions should be accurate, the organism used should be the same throughout, etc. These are not indictments of the test in careful hands, and where the tests on different disinfectants are made by the same individual. Still the results obtained by this test should not be taken for more than they pretend to be, the relative power of different bodies to carbolic acid under the same conditions. The application of such results to ordinary purposes must be made very cautiously. The "Lancet" Commission Test. — This is a modified Rideal-Walker test, in which the number of dilutions is increased up to nine, and the time periods correspondingly up to thirty minutes ; B. coli is used as the test organism ; MacConkey's bile-salt litmus glucose peptone water is used as the medium for subcultures instead of broth ; platinum spoons are used instead of loops, and the method of calcula- tion is different. The B. coli used is cultivated for twenty-four hours at SPECIAL EXAMINATIONS 381 370 C. in a broth made by mincing i lb. of fat-free bullock's- heart meat ; macerate it in cold water for two to three hours ; cook over a small gas flame for two to three hours more ; boil ; filter ; make up to a litre ; add 10 grm. each of NaCl and Witte's peptones ; and standardize to an acidity of 1-5 per cent to phenolphthalein. To obtain an emulsion from the culture, it is well shaken and then filtered through a double layer of Swedish filter-paper. The carbolic acid dilutions were made at first by taking ac. carbolic B.P. no grm. = 100 grm. pure phenol. Later, dry crystalline carbolic acid was taken as 100 per cent phenol. The dilutions were always freshly made, as it was found that otherwise they lost some of their germicidal power, even when kept corked and in the dark. The dis- infectant dilutions are made with distilled sterile water, and 5 c.c. of each are put in small glass specimen pots, 2-5 in. X f in., arranged in holes on a board. These pots are left uncovered during the experiment, and the results are not vitiated owing to the MacConkey's media used for the subculturing, inhibiting most other organisms than B. coli. Owing to the platinum spoon holding three times the amount in a standard loopful, 10 c.c. are used for the subculture medium. To assist in rapid work a special wheel has been devised, to hold the spoons, when not in use, in a sterilizing flame. Process. — All the apparatus being ready and to hand, the dilutions of the unknown disinfectant are " seeded," each getting a spoonful of the B. coli emulsion, and being well stirred. The seeding is begun at the strongest dilution and proceeds to the weakest. At the end of 2-5 minutes, a spoonful is removed from the first dilution seeded and added to a tubeful (10 c.c.) of MacConkey's broth, properly labelled. The same process is repeated with all the tubes, and is all gone over again after 5, 7-5, 10, 12-5, 15, 20, 25, and 30 minutes. The same procedure is followed with dilutions of carbolic acid. The subcultures are incubated for forty-eight hours at 370 C. A positive result is indicated by the medium turning red, and the formation of bubbles of gas. In some tubes this change will appear in twelve to fourteen 382 PUBLIC HEALTH BACTERIOLOGY hours as a purple tinge, becoming pink and then red in another two hours. The results are filled into a tabular form, with a plus sign for growth and a zero for no growth. The coefficient is thus calculated : The weakest dilution of the sample under test, giving no growth at 2-5 minutes, is divided by the weakest dilution of carbolic acting similarly in the same time ; the weakest dilution giving no growth at thirty minutes is divided by the same for carbolic acid ; the two results are averaged, and the average or mean is taken as the carbolic acid coefficient. Thus, if Disinfectant B gives no growth with 1-220 in 2 -5 minutes and with 1-340 in 30 minutes, and if the dilutions for carbolic acid are 1-110 and 1-180, then the coefficient would be : — (H» + Hi) - 2 or (2 + i-88) + 2 = 1-94. The Commission prefer to express their dilutions as percentages, and when this is done the mode of division is reversed ; that is, the weakest percentage (of carbolic, etc.) is divided by the weakest percentage of the test substance, etc. The above dilutions become for B, 0-454 per cent and 0-294 per cent, and for carbolic, 0-909 per cent and 0-555 per cent respectively. The coefficient therefore is (%m + %'5U) - 2 or (2 + 1-85) 4- 2 *» 1-92. The tem- perature of the room averaged about 620 to 670 F. By this method the coefficients ranged from 0-025 to 9-8 for the usual coal-tar disinfectants on the market. Applied to corrosive sublimate, the coefficient at 2-5 minutes was about 2000, and at 30 minutes about 6000, giving a mean of about 4000. Chloride of lime similarly gave figures of 45 and 93, or a mean of 69. For formalin the coefficient was o-6. Among the conclusions reached were the following : That results obtained in such bacteriological experiments, although giving a germicidal value to a disinfectant under the most favourable conditions, afford little indication of their germicidal value when used in practical disinfection. That much remains to be done in the solution of such problems (amongst others) as arise, due to : (1) The presence of foreign substances in the material to be dis- infected ; (2) The temperature at which the disinfecting SPECIAL EXAMINATIONS 383 process is carried on ; (3) The kind of water used for dilution — hard water, soft water, or sea water ; (4) The type of micro-organism that has to be dealt with ; (5) The nature of the substance to be disinfected and the character of its surface ; (6) The time to be allowed for the process. That only when the influence of such factors can be calculated will it be possible to modify any standard co- efficient figure, and thus to obtain data for the preparation of effective and economical dilutions for the practical problem of disinfection. The inquiry has shown that, so far as emulsions are concerned, those which contain the highest quantity of phenoloids in the finest state of division and having the least tendency to combine with albumins, lime, or other foreign substances in solution (and remain combined), will be found to be the most efficient disinfectants. The remarkable parallelism between the results of this inquiry (as shown in the figures for the coefficient) and the results of the independent chemical one, is very striking. The chemical commissioners discovered that if they subtracted the carbolic acid equivalent of the bromine absorbed by the percentage of phenoloids present, from the percentage of phenoloids present, and divided the difference by 3, the figures obtained in many instances are the same (or very nearly so) as those assigned as the carbolic acid co-efficient for the same substance by the bacteriological commissioners. In the exceptions the dis- infecting fluids did not form an emulsion with water, nor show Brownian movements. (See p. 142.) ^* = C.C. 3 Tables showing the results of the examination (on these principles) of the disinfectants in common use, are given in the Lancet for 1909, vol. ii. APPENDIX. REGULATIONS FOR THE DIPLOMA IN PUBLIC HEALTH. I. — The Council, having regard to the terms of Section 18 of the Local Government Act (1888) and of Section 54 of the Local Government (Scotland) Act (1889), and observing that under those sections special privilege is to be accorded to the holders of the diplomas granted under Section 21 of the Medical Act (1886), and therein described as Diplomas in Sanitary Science, Public Health, or State Medicine, thinks it essential to declare, with regard to its own future action under Section 21 of the Medical Act (1886), that it will not consider diplomas to " deserve recognition in the Medical Register " unless they have been granted under such conditions of education and examination as to ensure (in the judgment of the Council) the possession of a distinctively high proficiency, scientific and practical, in all the branches of study which concern the public health ; and the Council, in forming its judgment on such conditions of education and examination, will expect the following rules to have been observed : — Rule i. The curriculum for a Diploma in Sanitary Science, Public Health, or State Medicine shall extend over a period of not less than nine calendar months. Rule 2. Every candidate for a Diploma in Sanitary Science, Public Health, or State Medicine shall have produced satis- factory evidence that, after obtaining a registrable qualification, which should be registered before admission to examination for the diploma, he has received practical instruction in a laboratory or laboratories, British or foreign, approved by the licensing body granting the diploma in which Chemistry, Bacteriology, and the Pathology of the Diseases of Animals transmissible to Man are taught. Note. — The laboratory instruction shall cover a period of not less than four calendar months, and the candidate shall produce evidence that he has worked in the laboratory for at least 240 hours, of which not more than one-half shall be APPENDIX 385 devoted to practical chemistry. The laboratory course should be so arranged as to lay special stress on practical work which bears most directly on the duties of a medical officer of health. Rule 3. Every Candidate shall have produced satisfactory evidence — Either (1) that, after obtaining a registrable qualification, he has during six months been diligently engaged in acquiring a practical knowledge of the duties, routine and special, of public health administration, under the personal supervision of (a) In England and Wales, the medical officer of health of a county or of a single or combined sanitary district having a population of not less than 50,000, or a medical officer of health devoting his whole time to public health work ; or (b) In Scotland, a medical officer of health of a county or counties, or of one or more districts having a population of not less than 30,000 ; or (c) In Ireland, a medical superin- tendent officer of health of a district or districts having a population of not less than 30,000 ; or (d) In the British dominions outside the United Kingdom, a medical officer of health of a sanitary district having a population of not less than 30,000, who himself holds a registrable Diploma in Public Health ; or (e) A medical officer of health who is also a teacher in the department of public health of a recognized medical school ; or (/) A sanitary staff officer of the Royal Army Medical Corps having charge of an army corps, district, com- mand, or division, recognized for this purpose by the General Medical Council ; or (g) An assistant medical officer of health of a county or of a single sanitary district having a population of not less than 50,000, provided the medical officer of health of the county or district in question permits the assistant officer to give the necessary instruction and to issue certificates ; Or (2) That he has himself held for a period of not less than three years an appointment as medical officer of health of a sanitary district within the British Dominions, and having a population of not less than 15,000. Note 1. — The certificate for the purpose of Rule 3(1) must include testimony that the candidate has attended under the supervision of the person certifying on not less than 60 working days. Provided that if the candidate has : — (i) Pro- duced satisfactory evidence that he has attended a course or courses of instruction in sanitary law, vital statistics, epidemiology, school hygiene, and other subjects bearing on public health administration, given by a teacher or teachers 25 386 PUBLIC HEALTH BACTERIOLOGY in the department of public health of a recognized medical school ; or (ii) Produced evidence that he has been a resident medical officer in a hospital for infectious diseases containing not less than ioo beds, during a period of three months — the period during which he has been engaged in acquiring practical knowledge of his duties under this rule may be reduced to three months, to include an attendance on at least 30 working days. Note 2. — For the districts, commands, and divisions that have been recognized by the Council under Rule 3 (1) (/) see below. Rule 4. Every candidate shall have produced evidence that, after obtaining a registrable qualification, he has attended during three months at least twice weekly the practice of a hospital for infectious diseases at which candidate has received instruction in the methods of administration. Note 1. — Methods of administration shall include the methods of dealing with patients at their admission and discharge, as well as in the wards, and the medical super- intendence of the hospital generally. Note 2. — In the case of a medical officer of the Royal Army Medical Corps a certificate from a principal medical officer under whom he has served, stating that he has during a period of at least three months been diligently engaged in acquiring a practical knowledge of hospital administration in relation to infectious diseases, may be accepted as evidence under Rule 4. *„<* The Rules 2, 3, 4, as to study, shall not apply to medical practitioners registered, or entitled to be registered, on or before Jan. 1st, 1890. Rule 5. The examination shall have been conducted by examiners specially qualified ; it shall have extended over not less than four days, one of which shall have been devoted to practical work in a laboratory, and one to practical examina- tion in, and reporting on, subjects which fall within the duties of a medical officer of health, including those of a school medical officer. II. — The Council shall, from time to time, appoint an inspector or inspectors of examinations in public health, with special instructions to report to the Council whether the examination of each licensing body does or does not afford evidence, on the part of candidates passing such examination, APPENDIX 387 of a distinctly high proficiency, scientific and practical, in each and all of the branches of study which concern the public health. List of the districts and commands that have been recognized by the Council under Rule 3 (/) : — Aldershot. Salisbury Plain. Southern and South-Eastern Western. Dublin and Belfast. Cork. Chatham and Woolwich. Home. Eastern. North-Eastern and North- western. Scottish. Gibraltar Command. Malta Command. The following Indian Divi- sions, viz. : — 1st (Peshawar). 2nd (Rawalpindi). 3rd (Lahore). 5th (Mhow). 6th (Poona). 7th (Meerut). 8 th (Lucknow). 9th (Secunderabad). Burma. Quetta 388 PRESERVATIVES IN MILK AND CREAM. The Local Government Board, England, in February, 191 2, in the exercise of its powers under the Public Health Acts, drafted regulations prohibiting the use of preservatives in milk and denning the conditions under which preservatives may be used in cream. PRESERVATIVES IN MILK. Article III. : " 1. No person shall add, or order or permit any other person to add, any preservative substance to milk intended for sale for human consumption. 2. No person shall sell, or expose or offer for sale, or have in his possession for the purpose of sale, any milk to which any preservative substance has been added in contravention of subdivision (1) of this article." The expression " milk " includes separated, skimmed, condensed, and dried milk ; but as the traffic in condensed milk would be seriously impeded if the use of sugar were disallowed, it is provided that " neither cane nor beet sugar shall be regarded as a preservative or a thickening substance." PRESERVATIVES IN CREAM. " I. No person shall add, or order or permit any other person to add, (a) any thickening substance to cream or preserved cream ; {b) any preservative substance to cream containing less than 40 per cent by weight of milk fat ; (c) to cream containing 40 per cent or more by weight of milk fat any preservative substance other than (i.) boric acid, borax, or a mixture of these preservative substances, or (ii.) hydrogen peroxide, in amount not exceeding 0-1 per cent by weight, in any case in which the cream is intended for sale for human consumption. 2. No person shall sell, or expose or offer for sale, or have in his possession for the purpose of sale, any cream to which any thickening substance or any preservative substance has been added in contravention of the provisions of subdivision (1) of this article." Every seller of preserved cream will be required in every invoice, bill, advertisement, trade list, or other document which is used in connection with the sale of preserved cream, to describe the article as (a) pre- served cream (boracised), or (b) preserved cream (peroxidised), as the case may be. (This provision will come into force on APPENDIX 389 January i, 191 3.) Dealers in cream, preserved in a manner which does not contravene the above regulation, will be required, by means of labels on the receptacles, to declare that the cream is preserved and to state the name of the preservative. In this matter the regulations are precise, laying down in a schedule the size of the label, which varies according to the capacity of the receptacle, and prohibiting the attachment to any receptacle of a label bearing a trade description which would be likely to mislead a purchaser as to the utility of the preservative substance. In tea shops and other refreshment rooms the cream jugs will not be required to be labelled if in each room a conspicuous notice is affixed indicating that the cieam supplied is preserved cream, or if a statement to that effect is printed on the bills of fare. The regulations are made under the Public Health (Regulations as to Food) Act, 1907, and any person who wilfully neglects to carry out the regulations is liable to a penalty not exceeding ^100, and in the case of a continuing offence to a furthei penalty not exceeding £50 for every day during which the offence continues. The penalties for offences against the regulations are those for which provision is made by Sub- section (3) of Section 1 of the Public Health Act, 1896, but before the local authority institutes proceedings against any person they will be required to afford him an opportunity of explaining the circumstances in which any irregularity may have occurred. With the exception of the provision relating to the labelling of preserved cream, which takes effect on Jan. 1st, 191 3, the regulations take effect from June 1st, 191 2. 390 BOVINE AND HUMAN TYPES OF TUBERCLE BACILLI.* i. Smith Reaction. Theobald Smith, in 1896, first drew attention to the existence of two types oi tubercle bacilli in mammals, and in 1898 he published a systematic comparative study of bacilli isolated from man and cattle, and pointed out the differences between the two types as summarised on page 277. Pursuing the research further, he found that when grown in slightly acid glycerin broth, the two types give different reaction curves. Glycerin Broth. — The human type caused at first a lessening of the acidity of the medium until it became nearly but not quite neutral ; thereafter the acidity increased until it again approached or slightly exceeded the original reaction. The bovine type in the early stages of its growth rendered the medium less acid, neutral, or even alkaline ; and then it may so remain or become acid again, but never up to the original degree. The British Royal Commission for this test also used glycerin litmus milk (milk freed from cream, plus 5 per cent of glycerin, plus 5 per cent of 5 per cent watery solution of Merck's purified litmus). They found that all human viruses which grew vigorously on this medium at first caused an increase of alkalinity, and later acidity and clotting ; if the growth was less vigorous, acidity resulted without clotting. The bovine viruses which grew vigorously in the medium caused acidity finally, but never clotted the milk ; the poorly growing viruses left the milk alkaline. They concluded that the reaction curves can be so grouped as to form a scale of the final reactions with complete gradations from one type to the other. 2. Cultural Characters (p. 15). The results of our work have led to the conclusion that there is no constant qualitative cultural difference between * Abstracted from Vol. V. of " Collected Studies from the Research Laboratory, Department of Health, City of New York, 1910," by Park and Krumwiede, and others. APPENDIX 391 the human and bovine types of tubercle bacilli. Quantita- tively, and with respect to the effect of glycerin, however, there is a marked difference in the great majority of cultures, so great in fact that almost without exception the type can be determined from cultures alone. This difference is constant in one factor only, viz., amount and rapidity of growth in early cultures. In classifying our cultures according to this characteristic, we can broadly say that all bovine types of bacilli are dysgonic (sparse growth) and all human types of bacilli are eugonic (moderate or luxuriant growth). The question remains, What is the best medium for eliciting this difference ? Any medium used must fulfil certain conditions, i. It must be especially adapted for the growth of the eugonic viruses, so that the best possible growth is obtained. 2. It would be preferable if the growth of the dysgonic virus were somewhat retarded on the medium. This will widen the gap as far as possible. 3. The medium must be nearly uniform in its results ; that is, growth should not fluctuate with the different batches of medium used. We have found that glycerin egg is by far the best medium for this purpose. . . . Cobbett (Royal Commission) made comparisons of the two types of organisms on both serum and glycerin serum. While the human cultures were far more vigorous with the use of glycerin serum, the bovine cultures were either restrained, or if aided by its presence, the increase in growth was slight. This difference he found in early cultures to be of diagnostic value in the separation of the two types. We also noticed that primary cultures of the bovine type repeatedly failed on glycerin egg, whereas the primary cultures of the human type were usually markedly increased in luxuriance. The reading of Cobbett's results led us to adopt glycerin egg as the basic differential medium. Giving then the results in terms of glycerin, the following general conclusions may be drawn. (a). All cultures* growing luxuriantly on glycerin egg from the start are of the human type. (b). All cultures* growing sparsely, or even not at all, on * No direct cultures were attempted. Guinea-pigs were inoculated and killed in three to five weeks, and inoculations made from tuberculous lymph nodes and spleen on to plain egg media (Dorset), glycerin egg media (Lubenau), and glycerin potato. Intravenous inoculation into rabbits was used in confirmation of type of culture. If rabbit survived injection of 1 mgr. of culture for 60 days, then human type. 392 PUBLIC HEALTH BACTERIOLOGY glycerin egg in the first few generations are of the bovine type. Glycerin egg (Lubenau). Ten eggs are blown into a flask and 200 c.c. of 5 per cent glycerin bouillon (neutral or moderately alkaline) added. The further preparation is as for plain egg medium. 3. Results. Table of Series of Non-selected Cases of every Type of the Disease, showing Type of Bacillus Isolated. Adults Children Children Form of Tuberculosis 16 years and over 5-16 years 0-5 years Human Bovine Human Bovine Human Bovine Pulmonary 278 — 8 5 — Adenitis, cervical 9 — 19 8 6 12 Do. inguinal and axillary 1 — 4 — Abdominal 1 — 1 1 3 General (alimentary in origin) — — — — 1 1 General 2 — 1 — 12 4 ♦General + Meningitis — — - — — 18 1 Meningitis — — 1 — M 1 Bones and Joints 1 10 — 6 — Genito-urinary 3 I 1 — — — • Abscesses 1 435 296 1 45 9 62 22 297 54 84 4. Conclusions (Park and Krumwiede) loc. cit. p. 134. Tubercle bacilli as isolated from man fall into two groups. One of these groups is identical in all its characters with that found in cattle. That is, all tubercle bacilli from man and cattle fall into two groups, which have been designated the human and bovine types. Each type shows certain differences, the most important for separation being those culturally and in virulence. The great majority of cultures group themselves around two extremes, from which there are a few cultures showing variant characteristics. There is no overlapping of characteristics. The two types are probably different because of residence in different hosts over long periods of time, and as such are stable. The evidence in favour of rapid change of type is incomplete and inconclusive. * Includes 1 double infection : Mesenteric nodes gave human type, meningeal fluid gave bovine type. 393 INDEX PAGE PAGE A BIOGENESIS . . I48 Air, bacteriological examina- i\ Acarus sacchari, or sugar tion of 363 mite I09 — carbon monoxide in 69 Accelerated reaction 215 — estimation of C02 by Acetic acid in vinegar 124 Haldane's method . . 64 Achorion Schoenleinii 343 Hesse's method . . 65 Acid in beer, spirit indicatior L — — — Lunge and Zecken- value of 145 dorf's method 65 — permanganate in Tidy's — — — Pettenkofer's methoc 63 process 44 Scurfield's apparatus 66 Acid-fast bacilli in sections. 167 — examination of . . 62 — bacteria . . . . 163, 284 — ground 7i Acidimetry and alkalimetry 16 — moulds v. bacteria in 364 Acidity of beer 128 — noxious emanations in . . 67 — bread 100 — organisms found in 364 Actinomyces 286 — oxidizable and organic Actinomycosis 286 matter in 66 Active immunization 184 — oxygen in . . 69 Adams' process for fat in milli : 75 — ozone in . . 66 Adonite 106 — suspended matter in 69 Adulteration of butter 87 Albuminoid ammonia in water 43 — cereals 97 Alcohol in beer, direct distilla- — cocoa 119 tion 128 — coffee 113 Tabarie's method 128 — flour 99 — ethyl 132 — honey in — methyl 134 — milk, calculation in 76 — table, short 144 — mustard . . 112 Alcohol-fast bacteria 163 — pepper 112 Aldehydes in spirits 134 — tea ii5 Alexines . . . . 183, 203 Aerated bread 100 Alkalimetry and acidimetry 16 — waters, analysis of 53 — method (Hehner's) for Aerobic sporing bacilli 304 hardness in water 37 Afterdamp in mines . . 70 — sources of error in 17 Agar, beer-wort 344 Alkaline permanganate 43 — glucose 153 Alum in baking powders . . IOI — glycerin 153 — bread 100 — lactose 153 — flour 99 — litmus lactose 153 Aluminium process for nitrates — nutrient • 153 in water 50 — rat • 255 Amboceptor 203 — salt • 255 Ambulant plague 260 Agglutination . . . 191 Ammonia in water, albuminoid 43 — in cholera • 322 — — free and saline 40 — test in glanders . . • 253 — chloride, standard solution — in typhoid fever . . 234 of 4i Agglutinins 192 Amyl alcohols 134 Aggressins . . . . 17 7, 197 Amylum or starch 104 Agin B. coli . . • 357 Anaerobic sporing bacilli 304 394 INDEX PAGE Analyses of Glasgow sewage and effluents : . . . 61 — sewage and sewage effluents, tables of . . 56, 6 1 Autoclave sterilization . . 155 Autolysins .. .. .. 211 Avian tubercle bacilli 278, 291 Avogadro's law . . . . 14 •281 — of waters, table of 55 Analysis of aerated waters . . 53 Bacillary emulsion — chemical . . 5 Bacilli of the haemorrhagic- — colorimetric 5 septicaemic group — gravimetric 5 Bacillus acidi lactici (Hueppe) — of ice 53 (Leichmann) 242 — of mineral waters 53 — of acute conjunctivitis . . — of sewage from midden towns 56 — aerogenes capsulatus — volumetric 5 — of angular conjunctivitis — of water . . 19 — anthracoides interpretation of results 53 — anthracis . . Anaphylaxis . . . . 180 212 — of Bordet-Gengou — absolute . . 180 — botulinus — acquired . . 218 — Bulgaricus — classification 218 — of chicken cholera — to serum-globulin 217 — coli, agin . . — white of egg 215 communis Aniline oil-water stains 161 typical Annatto in butter 95 fermenting saccharose Anthrax bacillus . . 304 flaginac — orders 309 isolation of, from water Antianaphylaxis 215 sagin . . — to white of egg 217 search for, in water . . Antibody-producer 203 typical (British Com- Antigen 203 mittee) — by complement-fixation . . 210 typical (Houston) Antiplague serum 261 — diphtheria . . Antiseptic 135 — of Ducrey . . Antitoxic sera 189 — dysenteries . . Antitoxin, diphtheria 248 — enteritidis (Aertryck) Antituberculous sera 282 (Gaertner) Appendix, chemical . . 144 sporogenes — short alcohol table 144 — faecalis alcaligenes — table of degrees of spirit — fusiformis . . indications 146 — of glanders — — Glaisher's factors 144 — Hoffmanni spirit indication value — of human plague . . of acetic acid in beer 145 — influenzas . . weights of 1 eft. of — of Johne . . . . 28=5, water vapour 145 — of Klebs-Loeffler . . Arabinose 105 — of Koch -Weeks . . Arsenic in beer 129 — lactis aerogenes . . Arsenious solution, standard 135 — leprce Artificial immunity 184 — of malignant oedema Ascospores 33i — mallei Ash of milk 74 — megatherium Asparagine 106 — mucosus capsulatus Aspergillosis 336 — of M orax-Axenfeld Aspergillus 335 — mycoides . . — flavus 336 — ozaenae — glaucus 335 — paratyphosus — repens 336 — pestis . . . . ' . . — fumigatus 335 — phlegmonis emphysema- Atomic weights, table of 13 tosae 319 INDEX 395 PAGE PAGE Bacillus pneumonia . . 240 " Black death " . . 254, 260 — of quarter evil 318 Black rat 258 — of rabbit septicaemia 254 Blastomycoses 333 — radicosus . . 309 Bleaching of flour 99 — of rhinoscleroma . . 242 — powder 135 — of septic pleuropneumonia 254 Blood films 168 — smegmatis 285 ■ — serum, Loe frier's . . 246 — of soft chancre . . 245 medium 154 — subtilis 310 " Blowers " . . 70 — of swine plague . . 254 Boiled milk, detection of 79 — tetani 311 Borax in milk 80 and errata — tuberculosis 273 Bordet-Gengou bacillus 244 — typhosus . . 231 Bordet and Gengou reaction 204 — typi murium 236 Boric acid in butter . . 92 — vulgatus . . 310 milk 80 — of whooping cough 244 Botulus bacillus 316 — xerosis 247 Bottom yeast . . 127, 333 — of Zur Nedden 245 Bouillon filtre 281 Baking powders, composition Bovine tubercle bacilli 277, 289 ,390 of IOI — tuberculosis 293 definition of . . IOI Brandy 132 Bacterial activity, results of 172 Braxy 3i8 — poisons 175 Bread, manufacture of 99 — protein 175 — acidity of 100 Bacteriological examination of — aerated 100 air 363 Brewer's yeast 332 dust 366^ Brewing-waters 127 - — — milk Broth, " ghee " 256 sewage, etc. . . 365 — glycerin . . 390 soil 365 — nutrient I5i — — water . . . . 21, 345 ■ — standardization of its re- value of . . 54 action I5i — media, classified 151 Bromine absorption process Bacteriolysins 191 for phenol . . 141, 137 Bacterioscopic examination of Brown rat 258 water 347 Brownian movements in dis- Barley-sugar 103 infectants 140 Baryta water . . 10, 3s !, 6l Brucine test for nitrates 111 Bates' saccharometer 126 water 49 Bed bug 260 Buboes in rats 262 Beer 125 Bubonic plague 257 — acidity of . . 128 145 Buchanan's rat agar . . 255 — addenda . . I30 Buchner's tube 3ii — arsenic in 129 Buddeized milk 83 — wort . . . . 126, 128 Budding 331 agar 344 Bug, bed 260 Beers from starches, etc. 127 Butter, adulterations 87 Beeswax, melting-point of . . in — average composition of 87 Beet sugar 103 — bacteriology of . . 376 Benzoates in milk 82 — boric acid in .92 Bicarbonates in water, with — colouring matter in 95 carbonates 31 — curd or casein in . . 88 with free C02 32 — detection of starch in 94 Bile-salt, neutral-red, lactose — fat in 88 agar 154 — v . margarine 95 — glucose litmus peptone — polariscope test . . 94 water 153 — salt in 88 Black damp in mines 70 — saponification equivalent of 93 396 INDEX Butter, water in Butter-fat, detection of cotton seed oil in — detection of sesame oil in — fixed fatty acids in — iodine absorption of — refractive index of — sesame oil in — specific gravity of — valenta test for . . — volatile fatty acids in PAGE 88 94 94 9i 94 92 94 92 92 88 Cacao butter in cocoa Cadaverin Caffeine in coffee — tannate in tea infusions — in tea Calculation method for fat in milk Calmette's test Cane sugar analysis of — — in milk Capsulated bacilli Capsule staining Carbol-fuchsin (Ziehl-Neelsen) Carbol-glycerin-fuchsin Carbol-methylene-blue Carbolic acid, bromine ab sorption process 141, coefficient, formula for — — — relation to per cent of phenoloids tests for — powders Carbohydrate reactions (short table of) Carbohydrates, classification of — definition of — table of Carbon dioxide in water, as bicarbonate and car- bonate as free C02 — and bicarbonate Carbon monoxide in air Carbonates in water, with bicarbonates Carbonic acid gas in air, estimation of. . 63-66 Casein in butter . . . . 88 Cases of human tuberculosis 294 Centinormal solution, defini tion of . . Ceratophyllus unisus Cereals, adulterations in — analysis of 117 173 113 116 116 76 281 103 106 78 240 163 161 161 161 137 143 143 137 138 240 102 102 106 31 30 32 69 31 269 97 96 Cereals, animal and vegetable parasites in . . Cervical buboes in rats Chancroid bacillus . . Charbon symptomatique Cheese, analysis — average composition of Cheddar — bacteriology of . . — poisonous metals in rind of — ripening of — varieties of Chemical analysis of tubercle bacilli water, value of — examination of water — properties of tubercle bacillus — sterilization Chemiotaxis Chicory — in coffee China ink method . . Chloride of tin in sugar Chlorides in water . . Choke damp in mines Cholera carriers — red reaction Cider . . — vinegar Cimex lectularius Citric acid in lime juice Citrus limetta — limonum Classification of fungi Clotted cream C02 in air, Haldane's method Hesse's method Lunge and Zeckendorf method Pettenkofer's method Scurfield's apparatus Coal dust in mines . . — tar disinfectants . . Cobbett on portals of entry of tubercle bacilli — on glycerin media Cobra venom Coca Cocoa . . — nibs Coco-nut oil in butter estimation of melting point of — — Reichert figure of source of Cocos nucifera Coffee 262 245 318 96 96 376 96 95 95 5 274 54 , 22 293 155 182 113 114 328 109 24 70 322 321 132 123 260 120 120 119 170 84 64 65 65 63 66 70 140 279 391 178 119 117 117 89 91 118 90 118 118 113 INDEX 397 PAGE Coffee, detection of chicory in 114 Colon bacillus . . . . 230 Colon-typhoid-dysentery group 230 Colorimetric analysis . . 5 Colour formation by bacteria 158 — reduction by bacteria . . 159 Colouring matter in butter . . 95 sugar . . . . . . 109 wine . . . . . . 131 Columella . . . . . . 334 Comma bacillus . . . . 321 Committee of R.I. of P.H., report of . . . . 347 Complements . . . . 183, 203 Condensed milk . . . . 85 — skimmed milks . . . . 85 Coniferin . . . . . . 106 Copper in green peas .. 112 — sulphate . . . . . . 139 — — in bread . . . . 100 — in water, estimation of amount . . . . 28 — — presence of . . 25 Copper-zinc couple process for nitrates in water . . 51 Corn cockle in flour . . . . 99 Cornish cream . . . . 84 Cotton-seed oil in butter fat 94 Cow's milk, average com- position of . . 73, 86 Cow-wheat in flour . . . . 99 Cream, composition of . . 84 — fat in . . . . . . 84 — in milk . . . . . . 73 — preservatives in, new regu- lations.. .. .. 388 — of tartar in wine. . . . 130 Cresols.. .. .. .. 138 Crith, the . . . . . . 14 Cultivated yeasts . . . . 333 Cultural methods . . . . 155 — reactions . . . . . . 156 Curd in butter . . . . 88 Cutaneous plague . . . . 260 — test with tuberculin . . 280 Cytases . . . . 183, 203 Dalmarnock sewage and effluent . . . . 61 Dalmuir sewage and effluent 61 Danysz's virus . . . . 237 Darnel seeds in flour . . 99 Dauglish's system of bread- making . . . . 100 Decimal mode of dilution . . 367 Decinormal solution, defini- tion of . . . . 8 Demerara sugar . . . . 109 PAGE Denys bouillon nitre . . 281 Deodorant . . . . . . . 135 Deviation of the complement 211 Devonshire cream . . . . 84 Dextrin . . . . . . 105 Dextrose . . . . . . 104 Dhobie's itch . . . . . . 338 Diagnosis of anthrax . . 307 — plague ... . . . . 262 in China . . . . 269 Differentiation of B. typhosus from B. coli . . . . 238 — staphylococci . . . . 222 — streptococci . . . . 223 Diphenylamine test for ni- trates in water . . 50 Diphtheria antitoxin . . 248 — bacillus . . . . . . 246 Diplococcus pneumoniae . . 224 Diploma in Public Health, regulations . . . . 384 Dirt in milk . . . . . . 84 Disaccharids or sugars . . 102 Discontinuous sterilization . . 155 Disinfectants . . . . . . 135 — bacteriological examina- tion of . . . . 377 standardization of . . 378 — chemical examination of 135 — co-efficient of, inhibition of 378 — germicidal strengths of . . 378 — Lancet Commission test . . 380 — Rideal-Walker test . . 379 — standardization of, chemical 139 — in watery solutions . . 377 Disinfection . . . . . . 155 Disr/osal of dead in plague in China . . . . 271 Dorset's egg medium . . 275 Dried milk . . . . . . 83 Dried mother's milk, compo- sition of . . . . 86 Drigalski and Conradi's medium . . . . 239 Dry heat sterilization . . 155 — wine . . . • • • 130 Ducrey's bacillus . . . . 245 Dulcite . . . • . . 105 Dust, bacteriological exam- ination of . . . . 365 Dysentery bacillus . . . . 237 Earth bacillus . . . . 310 Eberth's bacillus . . . . 231 Egg medium, glycerin . . 391 — — Dorset's . . . . 275 Ehrlich's side-chain theory.. 202 — theory of immunity . . 199 398 INDEX Endo-enzyme 332 Endo's medium 239 Endotoxins 176 Enrichment method of HofY- man and Ficker 361 Enzymes 332 Ergot in flour 100 Erythrasma 338 Escherich's B. coli . . ' 230 Ethers, compound . . 133 Ethyl alcohol 132 Examination of milk 73 — pus. . 224 Excretion of tubercle bacilli in milk 298 External anthrax 304, 306 Extracellular toxins . . 176 Farcy.. .. .. .. 251 — buds . . . . . . 251 — order of 1907 . . . . 253 Fat in butter, Polenske pro- cess . . . . . . 90 Reichert-Wollny pro- cess 88 — cocoa . . . . . . 117 — coffee . . . . . . 114 — milk, Adams' process . . 75 calculation method . . 76 — — Gerber's process . . 75 Leffmann-Beam process 75 maceration process . . 76 Werner-Schmidt method 74 Fate of tubercle bacilli in tissues . . . . 297 Favus , 343 Feeding experiments with tubercle bacilli . . 297 Fehling's solution : compo- sition of . . . . 78 Pavy's modification of 97 Ferrous sulphate . . . . 137 Film, to make a . . . . 161 Filtration of immune serum 204 Final report of Royal Com- mission on tubercu- losis 288 Findlay on portals of entry of tubercle bacilli . . 279 Finings . . . . . . 127 Fire-damp in mines . . . . 70 Fixateur . . . . 199, 203 Fixation of blood films . . 169 — the complement . . . . 204 Fixed fatty acids in butter- fat . . . . . . 91 Flagella staining . . . . 165 Flaginac B. coli . . . . 357 PAGE Flaginac group of reactions 230, 356 Fleischwasser . . .. .. 152 Flexner group . . • . . 238 Flour, ergot in . . 100 Food infection with avian tubercle bacilli . . 300 bovine tubercle bacilli 301 — — human tubercle bacilli 300 Formaldehyde .. .. 136 — in milk . . . . 80, 81 Formalin in milk, proportions used . . . . . . 81 — quantitative test for . . 136 Formula for carbolic acid co-efficient . . . . 143 Forschammer process for organic matter in water 39, 44 Frambcesia . . . . . . 329 Fraenkel's pneumococcus . . 224 Frankland's method of air examination . . . . 363 to estimate organic matter in water . . 38 Free mineral acid, tests for.. 121 — and saline ammonia in water . . . . . . 40 Friedlaender's bacillus . . 240 Fructose . . . . . . 104 Fruit sugar . . . . . . 104 Fuchsin, carbol- . . . . 161 — carbol -glycerin .. .. 161 Fungi . . . . . . . . 170 — ringworm . . . . . . 341 Furfurol . . . . . . 133 Fusel oil, tests f or . . . . 134 Fusiform bacillus . . . . 247 Gaertner's bacillus. . .. 237 Galactose . . . . . . 104 Gases in mines, table of . . 70 — volume and density of . . 14 Gelatin, liquefaction of . . 159 — liquefying organisms, table of . . . . . . 160 — nutrient . . . . . . 152 — sugar media, Houston's.. 358 Gemmation . . . . . . 331 Gerber's process for fat in milk . . . . . . 75 Germicide . . . . . . 139 Ghee broth . . . . . . 256 Giemsa's stain . . . . 168 Gin . . . . . . . . 132 Ginger .. .. .. 112 Glaisher's factors, table of . . 144 Glanders .. .. .. 251 INDEX 399 Glanders bacillus — order of 1907 Glasgow sewage, analyses of 103, 104, PAGE 250 253 6l I09 •• 153 •• 152 I07 no III 103, 104, 109 rotatory Glucose — agar — broth — in cane sugar — golden syrup — honey — starch sugar — syrup, specific power . . . . . . no Glucoses or monosaccharids . . 102 Glutin in flour . . . . 98 Glycerin agar.. .. .. 153 — broth . . . . . . 390 — egg medium . . . . 391 — litmus milk . . . . 390 Glycogen . . . . . . 105 Golden syrup.. .. .. no — — specific rotatory power no Gonococcus . . . . . . 227 Gram's method for sections. . 167 of staining . . . . 162 Gram-negative organisms, table of . . . . 162 Gram-positive organisms, table of . . . . 162 Gravimetric analysis . . 5 Granule staining . . . . 166 Grape sugar . . . . . . 104 Greensands strata, effect on water . . . . 47, 55 Griess's test for nitrites in water . . . . . . 47 Ground air . . . . 71 — moisture . . . . . . 72 — water . . . . . . 72 Grueber's reaction . . . . 236 Guarana .. .. .. 119 Guinea-pig test for glanders 252 H^molysin-producing or- ganisms . . . . 161 Haemolysis . . . . 160, 192 Haffkine's vaccine for plague 261 — — for cholera . . . . 323 Haldane's method for estima- tion of C02 in air 64, 69 Han ta . . . . . . 264 Hardness in water, definition of 34 determination of by standard soap solution 35 — — estimation of by Hehner's method . . 37 total, temporary and permanent . . 37, 38 Harrison's indicator in Feh- ling's test . . . . 108 Hay bacillus .. .. .. 310 Head mould . . . . . . 334 Heavy oils in carbolic acid . . 138 Hehner's alkalimetry method to estimate hardness in water . . . . . . 37 — and Richmond's formula 76 — test for formaldehyde in milk . . . . . . 80 Hesse's method of air exam- ination. . . . . . 363 Heterolysins .. .. .. 211 High fermentation . . . . 333 High-pressure sterilization . . 155 Histricopsylla genus . . 269 Hoffmann's bacillus . . . . 247 Hoffman and Ficker's medium 239 Homogenized milk . . . . 83 Honey.. .. .. .. Ill — adulteration of . . .. in — specific rotatory power of 111 " Honey " growth . . . . 251 Honeydew .. .. .. Ill Houston's lemco medium . . 222 — method for water exam- ination.. .. .. 356 — sugar gelatin media . . 358 Human versus bovine tubercle bacilli . . . . 277, 390 - tubercle bacilli . . 277, 290 .. 293 294, 392 — tuberculosis table of cases of Humanized condensed milk — cow's milk Humus Hydrogen peroxide in milk Ice, analysis of Ilosvay's naphthylamine test for nitrites in water . . Immune body Immunity — absolute . . — acquired . . . . 181, — and anaphylaxis — artificial . . — natural — specific — theories of Immunization, active — passive — anthrax — tetanus — unit Incubator test for sewage and sewage effluents 85 83 7i 82 53 48 203 181 180 183 180 184 181 183 198 184 188 307 315 191 57 400 INDEX acids 85 111 India ink method Indian Plague Commission Indicators, classification of — neutral point — sensitiveness of . . Indigo process for nitrates in water Indol formation, tests for Infant foods, analysis of — — preparation of table of Infection — in children and adults — conditioned by — effects of . . — mode of action Infective disease, definition Influenza bacillus Inhalation of plague Inoculation of animals — plague Inosite or muscle sugar Insoluble fatty acids butter-fat — volatile fatty butter-fat Internal anthrax Interpretation of water analysis Intestinal plague Intracellular toxins Inulin Inunction test with tubercu- lin Inversion of saccharose Invert sugar . . syrup Invertase Iodine absorption of butter fat — test for formalin . . sulphites Iron in water, estimation of amount — — presence of causing taste . . tests for Isolation of B. anthracis from hairs — B. coli from water — sp. cholerae . . 323 — tetanus bacillus Isolysins Itch, dhobie's PAGE 328 259 IO II IO 52 159 86 86 87 173 295 174 174 174 173 243 257 169 257 106 91 90 304, 306 results of 53 260 176 105 281 103 103 no 332 94 136 121 28 29 125 307 353 1 324 3ii 211 338 PAGE Kefir 375 Kjeldahl's method for total nitrogen in sewage . . 59 organic matter in water 40, 46 total nitrogen in milk 78 Klebs-Loeffler bacillus . . 246 Koch's bacillary emulsion . . 281 — postulates.. .. .. 150 — steam sterilizer . . . . 155 — tuberculin.. .. 279, 281 Koch-Weeks bacillus . . 244 Kola .. .. .. ..119 Koppeschaar's process . . 137 Koumiss . . . . . . 375 103 Jenner's stain . . . . 167 Johne's bacillus . . 285, 372 Jorissen's test for formalde- hyde in milk . . . . 81 Lacmoid papers, use of Lactic acid bacillus . . Lactose agar — broth — in milk — or sugar of milk . . Lactose-nutrose-agar Laevulose . . Lamirasacsal streptococci Lanc^-acetone-baryta method Lancet Commission on Stand ardization of Disin fectants — bacteriological test — reports on plague in China Lead in' water, determination of amount presence of Leffmann-Beam process for fat in milk Leishman's stain Lemon juice Leprosy bacillus Letts and Blake process Leucocidin Leucocyte extract Leucocytosis Levaditi's method Light oils in carbolic acid Lime juice Lime in water, tests for Liquefaction of gelatin Litmus lactose agar . . — milk, glycerin — solution, reactions of — whey Local inspection of water supplies Lockjaw Loe filer's blood serum — medium — methylene-blue . . 12 242 153 152 77 104, 109 239 104 359 139 139 380 263 27 25 75 168 119 285 58 177 196 182 328 138 119 29, 30 159 153 3907 11 154 53 3ii 246 239 161 INDEX 401 Low fermentation " Lumpy jaw " Lupus Lymphocytosis PAGE 333 286 295 182 352 153 76 .. 286 •• 239 .. 124 •• 3i7 304, 306 • • 252 • • 252 .. 125 122 228-9 •• 332 104 344 56 105 105 no MacConkey's broth, reaction of certain bacteria with — media Maceration process for fat in milk Madura foot Malachite-green medium Malic acid in cider vinegar . . Malignant oedema bacillus — pustule Mallein — test Malt beer — vinegar Malta fever Maltase Maltose — media . . . . 341 Manchester Ship Canal, stand- ard for effluents " Manna " Mannite Maple syrup Magnesia in water, tests for 29, 30 Maragliano's serum . . . . 282 Margarine v. butter . . . . 95 Margarine in butter, calcula- tion of amount of . . 89 — fats, Reichert- Wollny figure of 89 Marmorek's serum . . . . 282 Marmot . . . . . . 264 Mate . . . . . . . . 119 McCrorie's flagella stain . . 165 M'Fadyean's test . . . . 308 Measuring of solutions . . 12 Meat extracts and essences.. 112 Media, bacteriological, classified 151 — gelatin sugar (Houston's) 358 Medium, Raulin's — Sabouraud's Megalosporon Mel depuratum B.P. . . Melitose or raffinose . . Meningococcus Metaphenylene-diamine solu tion Metchnikoff's bacteriotherapy — phagocytic theory — spirillum . . Methods of anaerobic culture — cultural Methyl alcohol 336 34i 343 in 105 226 47 374 198 324 310 155 134 PAGE Methyl-orange solution, re- actions of .. .. 11 Methylene-blue, carbol . . 161 — Loefner's . . . . . . 161 — reaction (in anthrax) . . 308 Metric system . . . . 14 — — factors for conversion of 15 imperial equivalents of 16 Micro-organism of syphilis . . 327 Micrococci . . . . . . 220 Micrococcus catarrhalis .. 228 — lanceolatus . . . . 224 — melitensis . . . . . . 228 — tetragenus . . . . 228 Microscopic characters of coffee and chicory .. .. 114 tea leaves .. .. 117 — examination of milk . . 79 Microspironema pallidum . . 327 Microsporon . . . . . . 343 — audouini . . . . . . 343 — furfur . . . . . . 337 — minutissimum . . . . 338 Midden towns sewage, analysis of 56 Milk, actinomyces in . . 370 — alcoholic fermentation of 375 — ash of . . . . . . 74 — B. enteritidis sporogenes in 368 — bacteriological examination 366 standards . . . . 367 — benzoates in . . . . 82 — blue 375 — boiled . . . . . . 79 — borax in . . 80 and errata — buddeized . . . . 83 — butter . . . . . . 375 — butyric acid fermentation of 374 — calculation of adulteration 76 — cane sugar in . . . . 78 — centuf legalization of . . 373 — condensed . . . . 85 — cream in . . . . . . 73 — dirt in . . . . . . 84 — diseases of . . . . 375 — dried . . . . . . 83 — enumeration of bacteria in 367 — examination of . . . . 73 — faecal organisms in . . 368 — fat in . . . . 74-76 — fermentations of . . . . 373 — " fore " . . . . . . 369 — glycerin litmus . . . . 390 — green 375 — homogenized . . . . 83 — humanized . . . . 83 condensed . . . . 85 402 INDEX PAGE Milk, Johne's bacillus in . . 372 — lactic acid fermentation of 373 — lactose in . . . . . . 77 — leucocyte test . . . . 373 — media . . . . 154, 390 — microscopic examination of 79 — " mid " . . . . . . 369 — nitrogen in . . 78 — pasteurized . . . . 83 — physical characters of . . 73 — preservatives in 79 and errata — — in, new regulations . . 388 — proteids in . . 79 ■ — reaction of • . . . 73 — red 375 — ropy 375 — slimy . . . . . . 375 — soapy 375 — solids not fat . . . . 76 — specific gravity of . . 73 — streptococci in . . . . 372 — " strippings " . . . . 369 — total solids of . . . . 73 — tubercle bacilli in . . 368 — deposit . . . . 370 — from tubercular udders . . 369 — yellow 375 Millinormal solution, defini- tion of . . . . . . 8 Mineral acid, tests for free . . 121 — waters, analysis of . . 53 Mines, table of gases in . . 70 Minimum lethal dose . . 190 Minute bacilli . . . . 243 Modes of study of bacteria . . 156 Modification of tubercle bacilli 278, 298 Moist heat sterilization . . 155 Moisture in bread . . . . 100 Molasses .. .. .. no Monosaccharids or glucoses. . 102 Morax-Axenfeld diplobacillus 245 Moro's test . . . . . . 281 Mother's milk, average com- position of . . 86 Moulds . . . . . . 334 — head 334 — knob 335 — pencil . . . . . . 336 — ringworm . . . . . . 341 - — and yeasts . . . . 330 Mucor mucedo . . . . 334 Muguet . . . . . . 340 Mulkowal outbreak (tetanus) 314 Mus decumanus . . . . 258 — rattus . . . . . . 258 Muscle sugar or inosite . . 106 Mustard, adulterations of . . 112 Mustard, composition of .. 112 Mycelium . . . . . . 334 Mycetoma . . . . . . 286 Mycomycetes . . . . 334 Mycoses . . . . . . 330 " Nail-head " growth . . 241 " Naked " bacteria . . . . 380 Naphthylamine solution . . 48 — test for nitrites in water . . 47 Natural anaphylaxis . . 218 — immunity . . . . . . 181 Negative phase . . . . 195 Nessler's solution, prepara- tion of . . . . . . 40 Nesslenzing, defined.. .. 27 Neutral oils in disinfectants 140, 141, 142 — point of indicators . . n Neutrality indicators . . 10 " New butter value " . . 91 New tuberculin . . . . 281 Nitrates in water, tests for . . 49 Nitrites in flour (from bleach- ing) 99 — and nitrates in water . . 47 — in water, tests for . . 47 Nitrogen in meat extracts . . 112 — milk 78 — peroxide, use of, in bleach- ing flour . . . . 99 Nitroso-indol reaction . . 159 Nocard's experiments . . 278 Non-standardized solutions.. 9 Normal diphtheria antitoxin 191 — solutions . . . . . . 7 definition of . . . . 8 Noxious emanations in air, detection of . . . . 67 Nursing of plague . . . . 263 Nutrient agar . . . . 153 — broth . . . . . . 151 — gelatin . . . . . . 152 Oidium albicans . . . . 340 Oil of theobroma .. .. 117 Old tuberculin . . . . 279 Ophthalmo-tuberculin reaction 281 Opsonic action . . . . 193 — co-efficient of extinction 194 — estimation, Leishman's method . . . . 193 Wright's method . . 194 — index . . . . . . 194 Opsonins . . . . 183, 193 Organic carbon by Frankland's method . . . . 38 INDEX 403 l' AGE Organic, matter in air - . . 66 water, estimation of.. 38 — — by Forschammer process 39, 44 — — — — Frankland's method . . 38 Kjeldahl's process 40, 46 — — — — Wanklyn's method 39, 40 — nitrogen by Frankland's method . . . . 38 ratio to albuminoid ammonia . . . . 55 Original gravity of beer wort 128 — tuberculin . . . . 279 Oxalic acid, standard solution of, for baryta 32, 63 carbonates . . 31 Oxidizable matter in air . . 66 Oxygen in air . . . . 69 — absorption by organic matter in water . . 44 — (dissolved) in water . . 33 Ozaena bacillus . . . . 241 Ozone in air . . . . . . 66 Pappenheim's stain . . . . 285 Paracolon bacilli . . . . 236 Paraffin-section staining . . 166 — wax, melting-point of .. in Paraguay tea.. .. .. 119 Parasites in cereals . . . . 98 Paratyphoid " A " . . . . 236 — "B" 236 — bacilli . . . . . . 236 Park & Krumwiede's research on tubercle bacilli — Tables of results 278, 392 Conclusions . . . . 390 Cultural characters . . 391 Passive immunization . . 188 Pasteurized milk . . . . 83 Pathogenic penicillia . . 337 — yeasts 333 Pathology of plague in China 268 Pavy-Fehling method for sugar estimation . . . . 97 Peas, tests for copper in . . 112 Peaty colour and test for lead 28 Penicillium . . . . . . 336 — glaucum . . . . . . 336 Pepper, adulteration of . . 112 — composition of .. .. 112 Peptone water .. .. 153 Percentage index . . . . 194 Permanganate of potash . . 136 Perry . . . . . . . . 132 Petri's method of air exam- tion Petruschky's litmus whey Pettenkofer's method for estimation of C02 in air Pfeiffer's phenomenon — reaction Phagocytic index Phagocytosis Phenol, bromine absorption process . . 141, — tests f or . . Phenol-sulphonic acid, prepara- tion of . . 10, 50 — test for nitrates in water 50 Phenolic bodies in disinfectants 140, 142 Phenoloids, relation of, to carbolic acid co-efficient Phenolphthalein solution, re- actions of Phenomenon of Arthus — Pfeiffer — Theobald Smith . . Phloroglucinol test for form- aldehyde in milk Phosphates in water, tests for 29, Phosphoric acid in cereals . Phycomycetes Physical characters of milk. Pinot's method for CI in bleaching powder Piquette Pityriasis versicolor Plague bacillus — in California — China summary of conclusions of .. symptoms of Egypt — Glasgow . . — Harbin — India — Manchuria — in marmots — in rats — spots — in squirrels — Suffolk Pneumobacillus Pneumococcus Pneumonic plague in China Poisonous metals cheese . . — — vinegar 271 265 259 254 266 260 263 264 258, 262, 269 260 255 254 240 224 259 263 rind of 26A 363 154 63 191 322 194 181 i37 i37 i43 11 214 191 214 30 97 334 73 135 131 337 254 255 263 96 124 404 INDEX PAGE Poisonous metals in water . . 25 qualitative tests . . 25 quantitative tests 26 Poisons, animal and vegetable 178 — ■ bacterial . . . . . . 175 Poivrette, in pepper.. .. 112 Polarimeter test for sugar . . 107 Polariscope test for butter . . 94 Polenske figure for fats . . 91 — process for butter-fat . . 90 Polychrome stains . . . . 167 Polymorphism . . . . 149 Polysaccharids or starches . . 102 Porges- Meier reaction . . 210 Portals of entry of tubercle bacilli . . . . . . 279 Positive phase . . . . 196 Post mortem of rat (plague) 262 Postulates, Koch's . . . . 150 Potassium permanganate,alka- line solution of . . 43 — — normal solution of . . 9 standard solution for oxygen absorption . . 45 Potato bacillus . . . . 310 — medium . . . . . . 154 Precipitation . . . . . . 192 Precipitins . . . . . . 192 Preservatives in beer . . 129 — cream . . 84 and errata new regulations . . 388 — milk . . 79 and errata new regulations . . 388 Presumptive B. coli test . . 352 Prevention of anthrax . . 309 Primary abdominal tuberculosis 293 — — — table of cases of . . 294 Products of bacterial activity 172 Proof spirit . . . . . . 133 Prophylaxis of plague . . 262 — tetanus . . . . . . 315 Proteids in milk . . . . 79 Proteinochrome formation by bacteria . . . . 159 Protozoa . . . . . . 171 Ptomaines . . . . . . 172 Public Health, regulations for Diploma in, . . . . 384 Pulex cheopis . . . . 257 — irritans . . . . . . 259 Pus, examination of . . . . 224 Putrescin . . . . . . 173 Qualitative chemical analysis 5 Quantitative chemical analysis 5 Quarantine in plague in China 270 Quarter evil . . . . . . 318 Quinine injections and tetanus 314 Raffinose or melitose . . 105 Rainfall and bacteria content 351 Rat agar . . . . . . 255 — buboes . . . . . . 262 — flea . . . . . . 257 Rats in China . . . . 269 Raulin's liquid medium . . 336 Rauschbrand . . . . . . 318 Raw River Thames water, bacteria in . . 350, 351 Reaction of milk . . . . 73 Reaction, Smith . . . . 390 Reactions of sewage bacteria, table of .. 354, 355 Rebipelagar . . . . . . 350 Recommendations of Tuber- culosis Commission 302-3 Reference terms, reply to . . 299 Refractive index of butter-fat 92 Regulations for Diploma in Public Health .. 384 Reichert-Wollny figures for butter and margarine fats 89 coco-nut fat . . . . 89 fish-oils . . . . 90 — — palm oil . . . . 90 — process for butter-fat . . 88 Reinsch's test for arsenic in beer . . . . . . 129 Relapsing fever . . . . 326 Rhinoscleroma bacillus . . 242 Richmond's test for boric acid in milk . . errata and 80 Rideal-Walker test . . . . 379 Ringworm fungi . . . . 341 table of . . . . 342 Romanowsky stain . . . . 167 Rosolic acid solution, reactions of .. .. .. 12 Rotatory power, specific . . 107 Royal Commission on Tuber- culosis . . . . 288 Rum . . . . . . . . 132 Saccharate of lime in cream 84 Saccharates or sucrates . . 103 Saccharin in beer . . . . 129 Saccharomyces albicans . . 340 — cerevisiae . . . . . . 322 — niger 333 — rosaceus . . . . . . 333 Saccharimeter test for sugars 107 Saccharose . . . . . . 103 — in cane sugar . . . . 107 — gravimetric estimation of 108 — volumetric estimation of 108 Sagin B. coli . . . . . . 357 INDEX 405 Salicin Salicylic acid in beer milk test for Sabouraud's medium Salt agar — in butter Salts with enlarged molecules Sampling of air for analysis — sewage and sewage effluent for analysis . . — water for analysis 21, Saponification equivalent of butter fat definition of . . — value, definition of — values and equivalents, table of Schiff's test for formaldehyde in milk Schizomycetes Sclavo's serum Search for B. coli in water . . — — tetani (wounds) Sedgwick and Tucker's method of air examination . . Seminormal solution, definition of Sensitiveness of indicators . . Separated milks Septicemic plague Sera, antitoxic — antituberculous Serum, anticholera . . — antidiphtheritic . . — antiplague — antitetanus — blood — diagnosis in plague — disease — Margliano's — Marmorek's — precipitation — Sclavo's . . — sickness — Sobernheim's — Yersin's ... Sesame oil in butter fat Sewage Commission, standards for effluents . . — effluents, bacteriological examination of — percentage purification of, see tables . . 56, 61 — and sewage effluents, Birmingham sewage.. 56 — — — examination of . . 57 — — — Glasgow analyses 61 PAGE I06 129 82 121 341 255 88 Sewage and sewage effluents, incubator test Kjeldahl's method for total nitrogen Letts and Blake process midden towns sewage — Staffordshire experi- ments standards in suspended solids in — — — table of analyses . . watercloset towns sewage Shake culture Shellfish Shiga- Kruse group Shiga's bacillus Smegma bacillus Smith reaction Soap solution, preparation and standardization of 35, 36 Sodium sulphite . . 121, 139 Soil, bacteriological examina tion of . . — organisms found in — temperature Soils, examination of Solids-not-fat, in milk Soluble cocoa. . — toxins Solution, standard arsenious Solutions, normal . . — standard Soor Sorbite Sorghum — juice Soxhlet apparatus, used in Adams' process . . 75 Specific gravity of butter-fat 92 milk . . . . . . 73 — immunity . . . . . . 183 to bacteria . . . . 185 toxins . . . . . . 185 — rotatory power . . . . 107 of dextrose .. 107 glucose syrup . . no — — — golden syrup . . no honey .. .. in — laevulose . . . . 107 365 — — — saccharose.. .. 107 Specimen analyses of waters 55 Spirilloses . . . . . . 326 Spirillum aquatilis (Gunther) 325 — cholera? asiaticas . . . . 321 — danubicus . . . . 325 — deneke . . . . . . 325 57 345 93 93 93 93 81 171 307 35i 316 364 10 85 260 189 282 323 248 261 315 154 261 212 282 282 192 307 212 307 261 94 56 57 59 58 56 56 56 57 56 56 157 376 238 237 285 390 365 365 72 7i 76 117 176 135 7 6 340 106 103 no 406 INDEX Spirillum of Finkler and Prior 325 Starch iodine test for nitrites — Massowah . . • 325 in water . . 47 — Metchnikovi • 324 — paste in cream 84 — Obermeieri 326 in honey in — phosphorescent . . • 325 — sugar or glucose 103, 104 109 — of relapsing fever . 326 — sugaring of by diastase . . 105 — tyrogenum • 325 Starches, beers from . . 127 Spirit indication, table of — examination of IOI acid values • 145 — morphology of IOI — — — values of . . 146 — or polysaccharids 102 — proof • 133 Steam in sterilization 155 Spirits.. • 132 Sterilization 155 Spirochaeta pallida . . • 327 Streak culture 157 — pertenuis . . • 329 Streptococci . . 222 — recurrentis . 326 — lamirasacsal 359 — refringens . . • 327 — in water 358 — Vincenti • 327 Streptococcus lacticus (Kruse) Spirochetes • 326 242, 374 Spirochetoses 326 — mucosus 226 Spontaneous generation 148 Subsoil 7i Sporangium 334 — water 72 Spore staining 164 Substance sensibilatrice 203 Sporing bacilli 304 Sucrose 103 Sporotrichosis 338 Sugar barley . . 103 Sporotrichum Beurmanni . 338 — beet 103 Spread of plague (reference) 263 — cane . . . . 103, 106 — — in China 266 — Demerara 109 Stab culture 157 — fruit 104 Stability in culture of bovine — grape . . 103, 104, 109 tubercle bacilli 290 — maple 103 human tubercle bacilli 291 — milk . . . . 104, 109 Staffordshire experiments in — mite, or Acarus sacchari 109 sewage treatment 56 — muscle 106 Staining reactions and methods 161 — palm 103 — by Romanowsky stains . 168 — starch 109 " Stalactite " growth 256 Sugars or disaccharids 102 Standard arsenious solution 135 Sugaring of starch by diastase 105 — ferrous sulphate . . 137 Sulphanilic acid solution . . 48 — nitrite solution for water Sulphates in water, tests for analysis 47 2C ■ 30 — soap solution, preparation Sulphites in milk 82 and standardization 35, 36 — tests f or . . 121 — solution of ammonium Sulphuretted hydrogen in chloride 41 wines . . 7o oxalic acid for baryta 32, 63 water 34 carbonates 3i Sulphuric acid in vinegar 124, 125 — — potassium permangan Sulphurous acid ... 121 139 ate for oxygen absorp Surface soil 7i tion 45 Suspended matter in air 69 — solutions 6 — solids in sewage effluents 57 Standards in sewage effluents 56 Sweet wine 130 Standardization of disinfect Syrup, golden no ants, Lancet Commis — invert sugar no sion 139 — maple no — sera 189 Staphylococci 220 Tabarie's method for alcohol Starch or amylum 104 in beer 128 — in butter . . 94 Table of anaerobes (sporing) 320 INDEX 407 PAGE Table of animal temperatures 170 — carbohydrates and allied substances . . . . 106 — characteristics of colon- typhoid group . . 240 — composition of infant foods 87 — degrees of spirit indication 146 — gelatin-liquefying organ- isms . . . . . . 160 — Glaisher's factors . . 144 — gram-positive and gram- negative organisms . . 162 — to illustrate Frankland's method, applied to water 39 — of principal ringworm fungi 342 — reactions of sewage bacteria 354, 355 — researches by Park and Krumwiede . . 278, 392 — saponification equivalents 93 — sewage and sewage efflu- ent analyses . . 56, 61 — short alcohol — of spirit indication value of acid in beer — water analyses . . — weights of 1 eft. of water vapour Tannin in tea Tarabagan — disease Tartaric acid, test for — — in wine Tatten-Thomson test for formaldehyde in milk Tea — fresh v. exhausted leaves Temperature in soil Temporary hardness in water Terms of reference, replies to Tetanolysin Tetanospasmin Tetanus — bacillus — toxin Theobromine in cocoa Theories of immunity Thrush Tidy's process for organic 144 145 55 145 116 264 264 122 131 81 115 117 72 37 299 314 314 3ii 3ii 314 118 198 340 matter in water Tinned peas Titration, definition of Top yeast Torula niger . . — rosea Torulae Total hardness in water 39 127 44 112 6 333 333 333 333 37, 38 Total secondary products . . — solids of milk Toxins . . — extracellular, true, or soluble . . — intracellular or endo- — nature of . . Transmission of plague Treponema pallidum — pertenue Trichophyton mentagrophytes — Sabouraudi — tonsurans Trommsdorff's leucocyte test True toxins Tubercle bacilli — — avian type . . 278, bovine type 277, 289, 390 — — human 277, 290, 390 . (Park and Krumwiede's researches) 277, 390 — — in fish . . — — in milk pus, faeces and tissues — — sputum urine . . Tuberculin, new — -O . . — original — -R .. — tests in cattle — vaccine therapy Tuberculosis in birds — Commission, recommenda tions . . . . 302 — in home cattle — horses — sheep — swine — various animals Turmeric solution, reactions of Typhoid bacillus Typical B. coli (British Com mittee) . . . . 349 (Houston) . . . . 356 — — — communis . . 356 Unicellular micro-organ- isms . . . . . . 170 Vaccination for anthrax . . 307 Vaccine, anticholera . . . . 323 — antigonococcal . . . . 228 — antiplague • . 261, 270 — antistaphylococcal . . 221 — antitubercle . . . . 282 — therapy . . . . . . 194 for typhoid fever . . 233 Valenta test for butter-fat . . 92 134 73 175 176 176 178 257 327 329 343 343 342 373 176 273 291 279 284 284 283 284 281 281 279 281 283 282 296 303 276 296 276 295 296 12 231 408 INDEX PAGE PAGE Vibrio choleras 321 Water analysis, nitrates . . 49 Vincent's angina 247, 327 nitrites . . . . 47 Vinegar 122 and nitrates . . 47 — cider 123 volatile solids . . 23 — malt 122 — — organic matter, various — white 123 methods of estimation 38 — wine 122 oxygen absorption . . 44 — wood 123 — — permanent or fixed Viscogen in cream . . 84 hardness . . 37, 38 Vitality in culture of avian tubercle bacilli . . 293 Voges-Proskauer reaction 238, 355 Volatile soluble fatty acids in butter fat . . ... 88 Volume and density of gases 14 Volumetric analysis . . . . 5 — — requirements for . . 6 — methods . . . . . . 6 Von Pirquet's test . . . . 280 Wanklyn's method for or- ganic matter in water 39, 40 Wassermann reaction . . 328 — test . . . . 205, 209 — — value of . . . . 209 Water analysis . . . . 19 albuminoid ammonia 43 arsenic, presence of . . 26 bacteriological exam- ination . . 21, 345 bicarbonates and car- bonates . . . . 31 chemical examination 20, 22 chlorine . . . . 24 collection of sample 21, 345 — — dissolved oxygen . . 33 solids . . . . 22 estimation of amount of copper in . . . . 28 ■ of iron in . . 28 lead in 27 zinc in 29 fixed solids . . . . 23 Forschammer process, for organic matter 39, 44 Frankland's method, for organic matter . . 38 — — free carbon dioxide . . 30 and bicarbonate 32 — and saline ammonia 40 hardness, definition of 34 determination of.. 35 interpretation of results 53 Kjeldahl's process, for organic matter 40, 46 lime and magnesia 29, 30 — — phosphates . . 29, 30 physical examination 20, 22 poisonous metals . . 25 — — presence of lead, copper, iron or zinc . . . . 25 qualitative tests for poisonous metals . . 25 quantitative tests for poisonous metals . . 26 reaction . . . . 22 — — specimen analyses . . 55 sulphates . . 29, 30 sulphuretted hydrogen 34 temporary and remov- able hardness.. .. 37 — — tin, presence of . . 26 total hardness 37, 38 solids . . . . 22 Wanklyn's method for organic matter 39, 40 zinc, presence of . . 26 — bacteria found in. . .. 345 — bacteriological examina- tion of . . . . 345 methods of examination 346 sampling . . . . 345 standards . . . . 346 — in butter 88 — of crystallization . . . . 8 — dilutions . . . . . . 346 — enumeration of bacteria in 349 — ground . . • . . . 72 — isolation of B. enteritidis sporogenes . . . . 360 typhosus . . . . 360 — — sp. choleras . . . . 324 — subsoil . . . . . . 72 — tables of bacteria in 350, 362 Watercloset towns sewage, analysis of . . . . 56 Watercress . . . . . . 376 Weigert's staining methods 166, 167 Weighing with the chemical balance . . . . 12 — and measuring, rules as to 12 Weights, atomic, table of . . 15 Werner -Schmidt process for fat in milk . . . . 74 INDEX 409 Wheat flour, analysis of Whey, litmus Whisky White damp in mines — vinegar " Whitening " of sugar Whooping-cough bacillus Widal's reaction : interpre tion of results in typhoid fever Wild yeasts . . Wine . . — " substitute . . — vinegar Wood spirit . . — vinegar Wool-sorter's disease 304, Wort, beer . . . . 126, agar . . Wright's vaccine therapy 194, I'AGF. PAGE 98 Xerosis bacillus 247 154 132 70 123 Yams Yeast in beer . . 127, 332 329 333 Yeasts.. 33i IO9 — cultivated. . 333 244 — and moulds 330 235 234 333 — pathogenic — wild Yersin's anti-plague serum . . 333 333 261 130 i*3i Zinc chloride . . 139 122 — in water, estimation of 134 amount 29 123 tests for 25 306 Zur Nedden's bacillus 245 128 Zygospore 335 344 Zymase 332 282 9 rM^ *